Thymidine kinase expressing natural killer cell lines and methods of use

ABSTRACT

This invention relates to a natural killer cell line termed NK-92. The invention provides a vector for transfecting a mammalian cell which includes a nucleic acid sequence encoding a cytokine that promotes the growth of NK-92. Additionally, the invention provides an NK-92 cell, or an NK-92 cell modified by transfection with a vector conferring advantageous properties, which is unable to proliferate and which preserves effective cytotoxic activity. The invention further provides a modified NK-92 cell that is transfected with a vector encoding a cytokine that promotes the growth of NK-92 cells. The cell secretes the cytokine upon being cultured under conditions that promote cytokine secretion, and furthermore secretes the cytokine in vivo upon being introduced into a mammal. In a significant embodiment, the cytokine is interleukin 2. The present invention also provides methods of purging cancer cells from a biological sample, of treating a cancer ex vivo in a mammal, and of treating a cancer in vivo in a mammal employing a natural killer cell, such as NK-92 itself, an NK-92 cell which is unable to proliferate and which preserves effective cytotoxic activity, or natural killer cells transfected with a vector encoding a cytokine.

RELATED APPLICATION

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/403,910, filed on Oct. 27, 1999, which was basedon, and claimed benefit of, U.S. Provisional Application Serial No.60/045,885, filed on Apr. 30, 1997.

FIELD OF THE INVENTION

[0002] This invention relates to natural killer cells and their use inthe treatment of pathologies related to cancer or viral infections.Specifically, a particular cell line, NK-92, and modifications thereof,are disclosed. These cells are shown to be highly effective in thetreatment of these pathologies.

BACKGROUND OF THE INVENTION

[0003] Certain cells of the immune system have cytotoxic activityagainst particular target cells. Cytotoxic T lymphocytes (CTLs) arespecifically directed to their targets via antigen-derived peptidesbound to MHC class I-specific markers. Natural killer (NK) cells,however, are not so restricted. NK cells, generally representing about10-15% of circulating lymphocytes, bind and kill target cells, includingvirus-infected cells and many malignant cells, nonspecifically withregard to antigen and without prior immune sensitization (Herberman etal., Science 214:24 (1981)). Killing of target cells occurs by inducingcell lysis. MHC class restriction likewise is not involved. In theseways the activity of NK cells differs from antigen-specific and MHCclass-specific T cells, such as cytotoxic T lymphocytes. Use of NK cellsin the immunotherapy of tumors and malignancies is suggested by theseproperties, since many tumors are MHC class I deficient and therefore donot attract CTL activity. Adhesion molecules may also be involved in thetargeting of NK cells; for example, it is observed that the Fcy receptor(CD16) is expressed on NK cells. NK cells are large granular lymphocyteswhich lack CD3, and in addition to CD16, also may express Leu19 (Lanieret al., J. Immunol. 136; 4480 (1986)).

[0004] NK cells are activated when exposed to cytokines such asinterleukin-2 (IL-2), IL-7, IL-12, and interferons (Alderson et al., J.Exp. Med. 172:577-587 (1990); Robertson et al., J. Exp. Med. 175:779-788(1992)). The resulting cells are called lymphokine activated killer(LAK) cells. The spectrum of target cells is altered in activated NKcells compared to nonactivated cells, although the mechanism of killingmay be identical or similar (Philips et al., J. Exp. Med. 164:814-825(1986)).

[0005] More generally, killing activity in the cells of the immunesystem may be induced by treating a population of cells, such asperipheral blood mononuclear cells (PBMCs), with lymphokines. Suchpreparations contain LAK cells. LAK cells may also be generated fromautologous samples of peripheral blood lymphocytes. LAK cells haveantitumor killing activity while having essentially no effect on normalcells. They appear to purge leukemia (Long et al., Transplantation46:433 (1988); Xhou et al., Proc. Am. Assoc. Cancer Res. 34:469 (1993;abstract)), lymphoma (Schmidt-Wolf et al., J. Exp. Med. 174: 139 (1991);Gambacorti-Passerini et al., Br. J. Haematol. 18:197 (1991)) andneuroblastoma (Ades et al., Clin. Immunol. Immunopathol. 46:150 (1988)).NK cells, activated NK cells, and LAK cells are distinguishable by theircell surface markers and by the identity of the target cells that theykill.

[0006] Activated and expanded (i.e., cultured to proliferate) NK cellsand LAK cells have been used in both ex vivo therapy and in vivotreatment in patients with advanced cancer. Some success with ex vivotherapy has been observed in bone marrow related diseases, such asleukemia, breast cancer and certain types of lymphoma. In vivo treatmentmay be directed toward these and other forms of cancer, includingmalignant melanoma and kidney cancer (Rosenberg et al., N. Engl. J. Med.316:889-897 (1987)). LAK cell treatment requires that the patient firstreceive IL-2, followed by leukophoresis and then an ex vivo incubationand culture of the harvested autologous blood cells in the presence ofIL-2 for a few days. The LAK cells must be reinfused along withrelatively high doses of IL-2 to complete the therapy. This purgingtreatment is expensive and can cause serious side effects. These includefluid retention, pulmonary edema, drop in blood pressure, and highfever. In some cases in which these side effects occur, intensive careunit management is required.

[0007] Purging techniques have been applied in other circumstances aswell. Cytotoxic drugs or monoclonal antibodies combined with complement,and toxins, may be administered in order to bring about remission. Insuch cases bone marrow or blood stem cells, purged to reduce the numberof residual leukemic cells present, have been infused back into thepatient after the drug treatment (Uckun et al., Blood 79:1094 (1992)).Gene marking studies have shown that unpurged bone marrow may contributeto relapse in patients presumed to be in remission (Brenner et al.,Lancet 341:85 (1993)). This suggests that some form of purging ofautologous marrow or blood prior to transplantation is necessary(Klingemann et al., Biol. Blood Marrow Transplant. 2:68-69 (1996)).

[0008] Recently, preclinical studies have also demonstrated promisingantitumor activity in vivo with a lethally irradiated, MHC-unrestricted,cytotoxic T-cell leukemic clone (TALL-104) (Cesano et al., CancerImmunol. Immunother. 40:139-151 (1995); Cesano et al., Blood 87:393-403(1996)). These cells were derived from leukemia T cell lines obtainedfrom patients having acute T lymphoblastic leukemias (ALL). They bearthe CD3 cell surface marker, but not the CD56 marker, in distinction toNK cells which have the converse immunophenotype (CD3⁻CD56⁺). Adoptivetransfer of these cells was able to eliminate human leukemic cell linesin xenografted severe combined immunodeficient (SCID) mice and to induceremissions of spontaneous lymphomas in dogs without producing T-cellleukemia in the animal models (Cesano et al. (1995); Cesano et al.(1996); Cesano et al., J. Clin. Invest. 94:1076-1084 (1994); Cesano etal., Cancer Res. 56:3021-3029 (1996)).

[0009] In spite of the advantageous properties of NK cells in killingtumor cells and virus-infected cells, they remain difficult to work withand to apply in immunotherapy. It is difficult to expand NK cells exvivo that maintain their tumor-targeting, tumoricidal, and viricidalcapabilities in vivo. This remains a major obstacle to their clinicaluse in adoptive cell immunotherapy (Melder et al., Cancer Research48:3461-3469 (1988); Stephen et al., Leuk. Lymphoma 377-399 (1992);Rosenberg et al., New Engl. J. Med. 316:889-897 (1987)). Studies of themechanisms whereby NK cells exert their tumoricidal and viricidaleffects are also limited by difficulties in enriching the NK cellfractions without compromising their biological functions and inobtaining pure NK cells that are not contaminated by T cells or otherimmune effector cells. In an attempt to overcome these problems, manyinvestigators have turned to the use of established NK-like cell linesto explore the mechanisms whereby target cells are susceptible tocytotoxic cells (Hercend et al., Nature 301:158-160 (1983); Yodoi etal., J. Immunol 134:1623-1630 (1985); Fernandez et al., Blood 67:925-930(1986); Robertson et al., Exp. Hematol. 24:406-415 (1996); Gong et al.,Leukemia 8:652-658 (1994)). NK cell lines described in earlier workcarry T lymphocyte-associated surface markers, in spite of the fact thatthey were developed from precursor cells depleted of T cells (Rosenberg,et al. (1987); Hercend, et al., (1983)).

[0010] There thus remains a need for a method of treating a pathologyrelated to cancer or a viral infection with a natural killer cell linethat maintains viability and therapeutic effectiveness against a varietyof tumor classes. This need encompasses therapeutic methods in whichsamples from a mammal are treated ex vivo with natural killer cells, aswell as methods of treatment of these pathologies with natural killercells in vivo in a mammal. There is also a need for a natural killercell line that maintains its own propensity for viability and cytolyticactivity by secreting a cytokine which fosters these properties. Therealso remains a need for such natural killer cell lines which aremodified to be more effective, convenient, and/or useful in treatment ofcancer and viral infection. It is the objective of this invention toprovide NK cells and methods that address these needs.

SUMMARY OF THE INVENTION

[0011] The cell line described by Gong et al. (1994), termed NK-92,proliferates in the presence of IL-2 and has high cytolytic activityagainst a variety of cancers. The present invention employs the NK-92cell line, as well as modified NK-92 cell lines, to provide cancertreatment and virus treatment systems. The invention also provides thevectors that transfect NK-92, as well as the modified NK-92 cells. Forpurposes of this invention and unless indicated otherwise, the term“NK-92” is intended to refer to the original NK-92 cell lines as well asthe modified NK-92 cell lines disclosed herein.

[0012] One aspect of the invention provides a vector for transfectingNK-92 cells, wherein the vector includes a nucleic acid sequenceencoding a protein that is either a cytokine which promotes the growthof the NK-92 cells, a cellular component responsive to an agent, acancer cell receptor molecule, or any combination of these proteins.When transfected with the vector, the NK-92 cells constitutively expressthe protein. In an important embodiment, the protein is the cytokineinterleukin 2. In especially important embodiments of this aspect of theinvention, the vectors are MFG-hIL-2 and pCEP4-LTRhIL-2. In additionalsignificant embodiments, the protein is a cellular component responsiveto an agent, such that when the vector transfects NK-92 cells and theagent is taken up by the cells, the cells are inactivated. In still moresignificant embodiments the agent is either acyclovir or gancyclovir.

[0013] A further embodiment of the invention provides a cell populationcontaining NK-92 cells that have been modified by a physical treatmentor by transfection with a vector.

[0014] In significant embodiments of this population, the physicaltreatment renders them non-proliferative yet does not significantlydiminish the cytotoxicity of the cells, and in particularly significantembodiments, the treatment is irradiation. In additional importantembodiments the cells have been transfected by a vector that encodes acytokine promoting the growth of the cells. The cells secrete thecytokine both upon being cultured under conditions that promote cytokinesecretion or in vivo upon being introduced into a mammal. Inparticularly important embodiments of this aspect of the invention, thecytokine is interleukin 2. In still further important embodiments, theNK-92 cells are the cells NK-92MI, modified by transfection with thevector MFG-hIL-2 encoding, and the cells NK-92CI modified bytransfection with the vector pCEP4-LTRhIL-2 encoding interleukin-2. TheNK-92MI and NK-92CI cell lines have been in the American Type CultureCollection under the designations CRL-2408 and CRL-2409, respectively.In additional important embodiments, the NK-92 cells are transfected bya vector including a sequence that encodes a cellular componentresponsive to an agent such that, when the NK-92 cell so transfectedtakes up the agent, the cell is inactivated. In particularly importantembodiments thereof, the agent is acyclovir or gancyclovir. In yetadditional embodiments, the cell population is transfected with a vectorencoding a cancer cell receptor molecule.

[0015] The present invention also provides a method of purging cellsrelated to a pathology from a biological sample including the steps of(i) obtaining a biological sample from a mammal that is suspected ofcontaining cells related to the pathology, and (ii) contacting thesample with a medium comprising NK-92 or modified NK-92 natural killercells, wherein the modified NK-92 cells have been modified by a physicaltreatment or by transfection with a vector. In significant embodimentsof this method, the pathology is a cancer, or is an infection by apathogenic virus such as human immunodeficiency virus (HIV),Epstein-Barr virus (EBV), cytomegalovirus (CMV), or herpes virus. Inadditional important embodiments, the modified NK-92 cells haveundergone a physical treatment that renders them non-proliferative, yetwhich does not significantly diminish their cytotoxicity, or have beentransfected with a vector, or they have been treated by any combinationof these modifications. In significant embodiments of this method, thevector encodes a cytokine that promotes the growth of the cells, aprotein that is responsive to an agent, a cancer cell receptor molecule,or a combination of these coding sequences. In a further embodiment, themedium also includes a cytokine that promotes the growth of the cells.The sample, once purged of cancer cells, may be further treated,including, for example, being returned to the mammal from which it wasobtained. In important embodiments of the method, the biological sampleis blood or bone marrow, the mammal is a human, and/or the naturalkiller cell is immobilized on a support.

[0016] The invention additionally provides a method of treating apathology ex vivo in a mammal including the steps of (i) obtaining abiological sample suspected of containing cells related to the pathologyfrom the mammal; (ii) contacting the biological sample with a mediumincluding natural killer cells, either NK-92 cells or modified NK-92cells that have been modified by a physical treatment or by transfectionwith a vector, thereby selectively destroying the cells related to thepathology in the sample and producing a purged sample, and (iii)returning the purged sample to the mammal. The pathology may be acancer, such as a leukemia, a lymphoma, or a multiple myeloma.Alternatively, the pathology may be infection by a pathogenic virus suchas HIV, EBV, CMV, or herpes. In this method the natural killer cells maybe NK-92 itself or modified NK-92 cells. Examples of such modified NK-92cells include those that have been modified by a physical treatment thatrenders them non-proliferative yet does not significantly diminish theircytotoxicity, and modification by transfection with a vector. The vectorencodes a cytokine that promotes the growth of the cells, or a proteinthat is responsive to an agent, or a cancer cell receptor molecule, orthe vector may include any combination of these modifications. Inimportant embodiments of this method, the biological sample is blood orbone marrow, the mammal is a human, and/or the natural killer cell isimmobilized on a support. In additional significant embodiments, themedium further includes a cytokine that promotes the growth of thecells, and/or the cancer is a leukemia, a lymphoma or a multiplemyeloma.

[0017] The present invention further provides a method of treating apathology in vivo in a mammal including the step of administering to themammal a medium comprising natural killer cells, either NK-92 cells orNK-92 cells that have been modified by a physical treatment that rendersthem non-proliferative yet does not significantly diminish theircytotoxicity, by treatment that inhibits expression of HLA antigens onthe NK-92 cell surface, or by transfection with a vector. The vectorencodes a cytokine that promotes the growth of the cells, or a proteinthat is responsive to an agent, or a cancer cell receptor molecule, orthey have been treated by any combination of these modifications. Inimportant embodiments, the pathology is a cancer, such as a leukemia, alymphoma, or a multiple myeloma. Alternatively, in important embodimentsthe pathology is infection by a pathogenic virus such as HIV, EBV, CMV,or herpes. Advantageous embodiments of this method include administeringthe cells intravenously to a human and administering a cytokine thatpromotes the growth of the cells to the mammal in conjunction withadministering the medium comprising the natural killer cell. The presentmethods are especially adapted for the treatment of leukemia, lymphomaor multiple myeloma.

[0018] In yet an additional embodiment of the in vivo method of treatingcancer, the NK-92 is modified by transfection with a vector comprisingan element responsive to an agent such that when the agent is taken upby the cell, the cell is inactivated. According to this method, anamount of the agent effective to inactivate the cell can be administeredto a mammal after a time sufficient for the natural killer cell to treatthe cancer has elapsed or at a time desirable to effectively end thetreatment. A significant aspect of this embodiment is one in which theagent is acyclovir or gancyclovir. Such transfected cells can, ineffect, be “turned off” as desired by administering the agent.

BRIEF DESCRIPTION OF THE DRAWING

[0019]FIG. 1. Cytotoxic activity of NK-92 against different leukemictarget cell lines tested in a 4 hour ⁵¹Cr release assay. The resultsrepresent the mean±the standard deviation (SD) for three replicateexperiments.

[0020]FIG. 2. Cytotoxicity of NK-92 after IL-2 deprivation. NK-92 cellswere cultured in enriched alpha medium (Myelocult™, StemCellTechnologies, Vancouver, BC) without IL-2. Cytotoxicity was measureddaily with the ⁵¹Cr-release assay against K562-neo′ or Daudi targetcells. The Figure shows results from one representative experiment atthe E:T ratio of 10:1.

[0021]FIG. 3. Effect of various doses of γ radiation on the cytolyticpotential of NK-92 cells. NK-92 cells were irradiated with a ¹³⁷Cssource using doses ranging from 200 to 1000 cGy. To allow for recovery,cells were left in medium containing IL-2 for 24 hours beforecytotoxicity was measured in a 4 hour ⁵¹Cr release assay against thetarget cell line K562.

[0022]FIG. 4. Survival curves of NK-92 cells after γ-irradiation. NK-92cells were irradiated with a γ ray source at doses of 300, 500, 1000,and 3000 cGy. Viability of NK-92 cells was determined by trypan bluestaining. The maximal achievable concentration of the non-irradiatedNK-92 cells in culture was about 1.5×10⁶/mL. The cells had to be fed toprevent overgrowth.

[0023]FIG. 5. Effect of γ-irradiation on the in vitro colony formationof NK-92 cells. NK-92 cells were cultured in agar-based mediumsupplemented with recombinant human IL-2 (rhIL-2).

[0024]FIG. 6. Effect of various radiation doses on the cytolyticpotential of NK-92 cells. NK-92 cells were γ-irradiated at doses of 300,500, 1000, and 3000 cGy. ⁵¹Cr-labeled leukemic target cells K562 (PanelA) and HL60 (Panel B) as well as two patient-derived leukemic samplesTA27 (Panel C) and BA25 (Panel D) were tested for susceptibility tocytolysis by irradiated and non-irradiated (NR) NK-92 cells. The resultsof 4 hr chromium release assays are expressed as 30% lytic units/10⁸effector cells.

[0025]FIG. 7. Selective killing of patient-derived leukemic cells byNK-92 cells. ⁵¹Cr-labeled leukemic target cells derived from 40 patients[9 acute myeloid leukemia (AML) cases, 11 chronic myeloid leukemia (CML)cases, 14 B-lineage-acute lymphoblastic leukemia (ALL) cases and 6 T-ALLcases] and T cell depleted normal bone marrow cells from 14 normaldonors were tested for susceptibility to cytolysis by NK-92 cells atfour different E:T ratios. The results of a 4 hr chromium release assayare expressed as 30% lytic units/10⁸ effector cells.

[0026]FIG. 8. In vitro (Panels A and B) and in vivo (Panels C and D)antileukemic efficacy of NK-92 cells against K562 and HL60 leukemias ascompared to human LAK cells and other effectors. ⁵¹Cr labeled K562(Panel A) and HL60 (Panel B) cells were tested for susceptibility tocytolysis by NK-92 cells in comparison with various known effector cells[LAK, NK (CD3⁻CD56⁺), and T cells (CD3⁺CD56⁻)] at indicated E:T ratiosin a 4 hr CRA assay. Results are means ±SD of three separate tests forNK-92 cells, and two tests of different donor-derived effectors for LAK,CD56⁺ and CD3⁺ cells. SCID mice were inoculated subcutaneously with K562cells (Panel C) or HL60 cells (Panel D) (5×10⁶ cells per mouse) alone orin combination with NK-92, LAK, or NK cells at a 4:1 E:T ratio. As ameasure of the tumor sizes, their surface areas were measured once aweek post inoculation (n=5).

[0027]FIG. 9 Antileukemic effect of NK-92 cells, allogeneic cytotoxic Tlymphocyte (CTL) cells and other effector cells against apatient-derived acute T lymphoblastic leukemia (T-ALL) determined invitro and in vivo. Panel A: In vitro specific killing of T-ALL (TA27)target cells by NK-92, CTL, and other effector cells, was determined bya 4 hr ⁵¹Cr-release assay using the indicated E:T ratios. Results aremeans ±SD of two or three separate tests. Panel B: SCID mice wereinoculated subcutaneously with TA27 cells (5×10⁶ each mouse) alone orco-inoculated with NK-92, CTL or other effector cells at a 4:1 E:Tratio. Recombinant human IL-2 (rhIL-2) was administered to the miceintraperitoneally for two weeks at the dose of 5×10⁴ U every other day.Leukemic tumor areas were measured once a week post inoculation (n=5).

[0028]FIG. 10. Survival of SCID mice bearing T-ALL (TA27) leukemiaco-inoculated with NK-92 cells as compared with co-inoculation withallogenic CTL or irradiated TALL-104 cells.

[0029]FIG. 11. Survival of SCID mice bearing T-ALL (TA27) aftertreatment with NK-92 cells. Mice received 5×10⁶ TA27 cellsintraperitoneally (I.P.). NK-92 cells (2×10⁷) were injected I.P. once,or 5 times (on days 1, 3, 5, 7 and 9), with or without the addition ofrhIL-2 every other day for two weeks.

[0030]FIG. 12. Survival of SCID mice bearing pre-B-ALL (BA31) aftertreatment with NK-92 cells. Mice received 5×10⁶ BA31 cells I.P. NK-92(2×10⁷) cells were injected I.P. for a total of 5 doses, on days 1, 3,5, 7 and 9. Mice in the indicated groups received rhIL-2 every other dayfor two weeks.

[0031]FIG. 13. Survival of SCID mice bearing human AML (MA26) aftertreatment with NK-92 cells. Mice received 5×10⁶ MA26 leukemia cells I.P.NK-92 (2×10⁷) cells were injected I.P. on days 1, 3, 5, 7 and 9 for atotal of five doses. Mice in the indicated groups received rhIL-2 everyother day for two weeks.

[0032]FIG. 14. Diagrammatic map of plasmid MFG-hIL-2.

[0033]FIG. 15. Diagrammatic map of plasmid pCEP4-LTR.hIL-2.

[0034]FIG. 16. PCR analysis of NK-92, NK-92MI and NK-92CI for human IL-2cDNA. DNA isolated from the parental NK-92 and from the NK-92MI andNK-92CI transfectants was subjected to PCR analysis with primersflanking the first exon of the human IL-2 gene. PCR products wereresolved on a 2% agarose gel, stained with ethidium bromide and viewedon a UV Transilluminator (Panel A). DNA was transferred to a nylonmembrane and analyzed by Southern blot analysis with a radiolabelledprobe for the hIL-2 gene (Panel B).

[0035]FIG. 17. Northern blot analysis of cytokine expression in NK-92,NK-92MI and NK-92CI. RNA samples isolated from the parental andtransfected cell lines were separated by agarose gel electrophoresisblotted to nylon membrane by capillary transfer and hybridized withprobes for human IL-2 (Panel A) and TNF-α (Panel B).

[0036]FIG. 18. Cytotoxicity of NK-92, NK-92MI and NK-92CI against K562and Raji target cells. The cytotoxic activities of the IL-2transfectants were compared to that of the parental cell line. NK cellswere mixed with ⁵¹Cr-labeled K562 (Panel A) or Raji (Panel B) cells ateffector:target ratios of 1:1, 5:1, 10:1 and 20:1 for a 4 hour chromiumrelease assay. The cytotoxicities of NK-92 (), NK-92MI (▴) and NK-92CI(▪) are shown.

[0037]FIG. 19. Effect of NK-92 MI and NK-92CI on hematopoieticprogenitors. To assay the effect of the NK-92 cells on normalhematopoietic progenitors, a clonogenic assay was performed. NormalPBMCs were incubated with irradiated NK-92MI or NK-92CI at variousNK:PBMC ratios ranging from 1:1 to 1:1000 for 48 hours. The cells wereplated in methylcellulose at concentrations to give 10-100 colonies perdish after 14 days. Clonogenic output of PBMCs incubated with NK-92MI(white bars) and NK-92CI (gray bars) is expressed as either total numberof colonies or subclassified on the basis of colony type (BFU-E, CFU-GMand CFU-GEMM).

[0038]FIG. 20. Effect of irradiation on NK-92, NK-92MI and NK-92CIproliferation and viability. To assess the effect of irradiation on theparental and transfected NK-92 cells, cells were exposed to 0, 500,1,000, 1,500, and 2,000 cGy doses of radiation and assayed forproliferation by a standard ³H-thymidine incorporation assay. Panel A:Proliferation of NK-92 (), NK-92MI (▴) and NK-92CI (▪) is expressed asa percentage of control (unirradiated cells). Panel B: Cells wereexposed to 0, 250, 500, 1,000, and 2,000 cGy of irradiation and assessedby trypan blue exclusion for viability after 24 (black bars), 48 (graybars) and 72 hours (white bars).

[0039]FIG. 21. Effect of irradiation on NK-92, NK-92MI and NK-92CIcytotoxicity. To assess the effect of irradiation on cytotoxicity of theNK cells, NK-92, NK-92MI and NK-92CI were irradiated at 0, 1,000, and2,000 cGy and tested after three days for cytotoxicity against K562(Panel A) and Raji (Panel B) cells.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention relates to methods of treating a biologicalsample or a mammal suspected of having a pathology such as a cancer oran infection by a virus. Certain natural killer cells which arecytolytic for the cells affected by the pathology are employed. Thetreatment results in significant diminution of the number, or, in somecases, the elimination, of malignant or cancerous cells, orvirus-infected cells, in the sample or mammal. The natural killer cellsof this invention are designated NK-92 cells and include certain treatedor transfected modifications of NK-92 cells. These cells are highlyeffective in purging cancer cells ex vivo and in destroying cancer cellsin vivo.

[0041] As used in the present invention, “cytotoxic T lymphocytes” (CTL)relate to immune cells which kill antigen-specific target cells. CTL areMHC class I-restricted. As used in the present invention, lymphokineactivated killer (LAK) cells relate to cells of the immune system thathave antitumor killing activity. They are obtained from a population ofcells, such as peripheral blood mononuclear cells, upon activation bytreatment with lymphokines. LAK cells have essentially no effect onnormal cells.

[0042] As used to describe the present invention, “natural killer (NK)cells” are cells of the immune system that kill target cells in theabsence of a specific antigenic stimulus, and without restrictionaccording to MHC class. Target cells may be tumor cells or cellsharboring viruses. NK cells are characterized by the presence of CD56and the absence of CD3 surface markers. The present invention is basedon an immortal NK cell line, NK-92, originally obtained from a patienthaving non-Hodgkin's lymphoma. As used to describe the presentinvention, a modified NK-92 cell is an NK-92 cell which has been furthertreated to endow it with properties not found in the parent from whichit is derived. Such treatments include, for example, physicaltreatments, chemical and/or biological treatments, and the like. Thetreatments confer properties upon the modified NK-92 cells that renderthem more advantageous for the purposes of the invention.

[0043] As used to describe the present invention, the terms “cytotoxic”and “cytolytic”, when used to describe the activity of effector cellssuch as NK cells, are intended to be synonymous. In general, cytotoxicactivity relates to killing of target cells by any of a variety ofbiological, biochemical, or biophysical mechanisms. Cytolysis refersmore specifically to activity in which the effector lyses the plasmamembrane of the target cell, thereby destroying its physical integrity.This results in the killing of the target cell. Without wishing to bebound by theory, it is believed that the cytotoxic effect of NK cells isdue to cytolysis.

[0044] As used to describe the present invention, “target cells” are thecells that are killed by the cytotoxic activity of the NK cells of theinvention. These include in particular cells that are malignant orotherwise derived from a cancer, and cells that are infected bypathogenic viruses such as HIV, EBV, CMV, or herpes.

[0045] As used to describe the present invention, “purging” relates tokilling of target cells by effector cells such as NK cells ex vivo. Thetarget cells may be included in a biological sample obtained from amammal believed to be suffering from a pathology related to the presenceof the target cell in the sample. The pathology may be a cancer ormalignancy due to tumor cells in the sample, and may be treated bypurging the sample of the tumor cells and returning the sample to thebody of the mammal.

[0046] As used to describe the present invention, “inactivation” of theNK-92 cells renders them incapable of growth and/or their normalfunction, in particular, their cytotoxic activity. Inactivation may alsorelate to the death of the NK-92 cells. It is envisioned that the NK-92cells may be inactivated after they have effectively purged an ex vivosample of cells related to a pathology in a therapeutic application, orafter they have resided within the body of a mammal a sufficient periodof time to effectively kill many or all target cells residing within thebody. Inactivation may be induced, by way of nonlimiting example, byadministering an inactivating agent to which the NK-92 cells aresensitive.

[0047] As used herein, a “vector” relates to a nucleic acid whichfunctions to incorporate a particular nucleic acid segment, such as asequence encoding a particular gene, into a cell. In most cases, thecell does not naturally contain the gene, so that the particular genebeing incorporated is a heterologous gene. A vector may includeadditional functional elements that direct and/or regulate transcriptionof the inserted gene or fragment. The regulatory sequence is operablypositioned with respect to the protein-encoding sequence such that, whenthe vector is introduced into a suitable host cell and the regulatorysequence exerts its effect, the protein is expressed. Regulatorysequences may include, by way of non-limiting example, a promoter,regions upstream or downstream of the promoter such as enhancers thatmay regulate the transcriptional activity of the promoter, and an originof replication. A vector may additionally include appropriaterestriction sites, antibiotic resistance or other markers for selectionof vector containing cells, RNA splice junctions, a transcriptiontermination region, and so forth.

[0048] As used to describe the present invention, “cancer”, “tumor”, and“malignancy” all relate equivalently to a hyperplasia of a tissue ororgan. If the tissue is a part of the lymphatic or immune system,malignant cells may include non-solid tumors of circulating cells.Malignancies of other tissues or organs may produce solid tumors. Ingeneral, the methods of the present invention may be used in thetreatment of lymphatic cells, circulating immune cells, and solidtumors.

[0049] As used to describe the present invention, a “pathogenic virus”is a virus causing disease in a host. The pathogenic virus infects cellsof the host animal and the consequence of such infection is adeterioration in the health of the host. Pathogenic viruses envisionedby the present invention include, but are not limited to, HIV, EBV, CMV,and herpes.

[0050] Natural Killer Cell NK-92. The NK-92 cell line has been describedby Gong et al. (1 994). It is found to exhibit the CD56^(bright), CD2,CD7, CD11a, CD28, CD45, and CD54 surface markers. It furthermore doesnot display the CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20,CD23, and CD34 markers. Growth of NK-92 cells in culture is dependentupon the presence of recombinant interleukin 2 (rIL-2), with a dose aslow as 10 IU/mL being sufficient to maintain proliferation. IL-7 andIL-12 do not support long-term growth, nor do other cytokines tested,including IL-1α, IL-6, tumor necrosis factor α, interferon α, andinterferon γ. NK-92 is highly effective in killing certain tumor cells,such as K562 (erythroleukemia) and Daudi (Burkitt lymphoma) cells, forit has high cytotoxicity even at a low effector:target (E:T) ratio of1:1 (Gong et al. (1994)). In addition, NK-92 cells have high cytotoxicactivity against 8E5 cells, which are infected with HIV and produce HIVvirions. NK-92 cells are deposited with the American Type CultureCollection, designation CRL-2407.

[0051] NK-92 cells are readily maintained in culture medium, such asenriched alpha minimum essential medium (MEM; Sigma Chemical Co., St.Louis, Mo.) supplemented with fetal calf serum (for example, at 12.5%;Sigma Chemical Co., St. Louis, Mo.), and horse serum (for example, at12.5%; Sigma Chemical Co., St. Louis, Mo.). Initially, 10⁻⁶ Mhydrocortisone is required, but in subsequent passages it is found thathydrocortisone may be omitted. In addition, IL-2, such as recombinanthuman IL-2 (500 U/mL; Chiron, Emeryville, Calif.), is required forlong-term growth. When suspension cultures are maintained in thisfashion with semiweekly changes of medium, the cells exhibit a doublingtime of about 24 h.

[0052] NK-92 cells in vitro demonstrate lytic activity against a broadrange of malignant target cells. These include cell lines derived fromcirculating target cells such as acute and chronic lymphoblastic andmyelogenous leukemia, lymphoma, myeloma, melanoma, as well as cells fromsolid tumors such as prostate cancer, neuroblastoma, and breast cancercell lines. This effect is observed even at very low effector:targetratios. This lysis is superior to cytotoxicity obtained from normalperipheral blood mononuclear cells stimulated for four days with IL-2.

[0053] Vector for transfecting mammalian cells to produce cytokine. Thepresent invention provides NK-92 cells which have been modified bytransfection with a vector that directs the secretion of a cytokine,such as IL-2. In order that NK-92 cells maintain long-term growth andcytolytic function, they generally must be supplied with IL-2. A vectorencoding the gene for human IL-2, and which also contains a controlelement directing the synthesis of the IL-2 gene product is therefore ofgreat utility in the invention. NK-92 cells bearing such a vectorsecrete the IL-2 needed for cytolytic activity in a therapeutic setting;thus, IL-2 from an exogenous source is not required. The control elementis one which directs the synthesis of IL-2 as a constitutive product,i.e., one that is not dependent upon induction. Methods for constructingand employing vectors are described in general terms in “CurrentProtocols in Molecular Biology”, Ausubel et al., John Wiley and Sons,New York (1987, updated quarterly), and “Molecular Cloning: A LaboratoryManual 2nd Ed.”, Sambrook, Fritsch and Maniatis, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989), which are incorporatedherein by reference.

[0054] Modified NK-92 transfected to produce cytokine, and method oftransfecting. Modified NK-92 cells that secrete a cytokine may beprepared by inserting a vector that directs the synthesis and secretionof the cytokine into the cells. In important aspects of the invention,the cytokine is IL-2. Methods of introducing a vector into a mammaliancell are well known to workers of ordinary skill in molecular biologyand cellular immunology, and are described in Ausubel et al. (1987,updated quarterly) and Sambrook et al. (1989). The vectors encoding thecytokine encompass as well control elements that lead to constitutivesynthesis of the cytokine when incorporated into the NK92 cells.

[0055] When cultured under appropriate conditions that promote cytokinesecretion the transfected NK-92 cells secrete IL-2 or other cytokine.Since the vector directs constitutive synthesis of the cytokine,nutrient cultures in which the NK-92 cells are known to grow and toexhibit their normal cytolytic function are sufficient for thetransfected cells to secrete the cytokine. For the same reason, thetransfected cells secrete the cytokine in vivo when they are introducedwithin the body of a mammal.

[0056] NK-92 cells transfected with a vector that directs secretion of acytokine such as IL-2 are useful in the ex vivo treatment of abiological sample drawn from a mammal which is suspected of containingmalignant cells. By treating the malignant cells with these modifiedNK-92 cells, the need for applying exogenous IL-2 or other cytokine isobviated. These modified NK-92 cells are useful, for the same reasons,in the in vivo treatment of a mammal suffering from a malignancy. Themodified NK-92 cells exert their cytolytic effect against the malignantcells when introduced into the body of the mammal. Examples of suchcells in the present invention are designated NK-92MI and NK-92CI.

[0057] NK-92 cells that are cytolytic but not capable of proliferation.An additional modified NK-92 cell of the invention is one that has beentreated in such a way that it is no longer able to proliferate, yetwhose cytotoxic activity is preserved. One way of achieving this stateis by γ irradiation. Additional forms of radiation, including, forexample, ultraviolet radiation, may be employed. Suitable sources to usefor this purpose include, for example, a ¹³⁷Cs source (Cis-US, Bedford,Mass.; Gammacell 40, Atomic Energy of Canada Ltd., Canada).Additionally, proliferative activity may be abrogated by treatment withchemical agents which inhibit DNA synthesis. An example of such an agentis mitomycin C.

[0058] Vector for transfecting NK-92 with an element responsive to aninactivating agent. The NK-92 cells may also be modified by transfectionwith a vector such that, when the cell takes up a specific agent, thecell is inactivated. The vector includes a sequence that encodes acellular component responsive to the agent, such that when the vectortransfects a cell and the agent is taken up by the cell, the cell isinactivated. In preferred embodiments, the agent is acyclovir organcyclovir. The vector also contains a control element directing thesynthesis of the cellular component as a constitutive product.

[0059] The NK-92 cell transfected with the vector described in thepreceding paragraph maintains its characteristic growth and cytolyticactivity in the absence of the agent. At a point in time, for example,when an ex vivo sample has been purged of malignant cells by the actionof the NK-92 cells, or when the NK-92 cells administered in vivo haveeffectively exerted their cytolytic activity within a mammalian body, orwhen it desired that the treatment be stopped for any reason, the agentmay be administered. The agent interacts with the cellular componentsensitive to the agent encoded in the vector. The interaction of theagent with the cellular component induces the inactivation of the NK-92cells. Inactivation may range from loss of characteristic cytolyticfunction to death of the cells.

[0060] This property of the modified NK-92 cells is significant becausethe parent NK-92 cells are derived from a tumor cell line that maycontinue propagating in a sample reintroduced into a mammal after exvivo therapy, or in vivo when so administered. It is therefore importantto ablate the cells after they have carried out their therapeuticfunction. Rendering the cells sensitive to an agent, such as acycloviror gancyclovir, is an advantageous way of achieving this objective.

[0061] Vector for transfecting NK-92 with an altered HLA cell surfacemolecule. The HLA cell surface protein, involved in presenting antigensto other cells of the immune system, includes a non-immunospecificsubunit, the protein β₂-microglobulin. If this protein is altered ormutated, the HLA protein loses its affinity for the T-cell receptor towhich it ordinarily binds. The β₂-microglobulin gene in NK-92 cells ofthe invention may be mutated by site specific mutagenesis in order totransform its properties in this way. The result is an NK-92 cell whichno longer has a high affinity for T-cell receptors. As a result, theNK-92 cell modified in this way remains within the host organism for alonger period of time, rather than being eliminated by the action of thehost's cellular immune response.

[0062] Vector for transfecting NK-92 with a gene encoding a cancer cellreceptor molecule. The NK-92 cells may also be modified by transfectionwith a vector such that the cells constitutively express a receptor fora cancer cell. Cancer cells express cell surface molecules that areidiosyncratic for the origin of the cancer, and frequently are alsoidiosyncratic for the individual host. The CTL population in suchdiseased patients may have been activated by exposure to the cells ofthe growing cancer. Such activated CTL express cell surface proteinsthat are specific for, or target, the cells of the cancer. These CTL maybe isolated, the gene for the targeting receptor identified, isolated,and transfected into the NK-92 cells of the invention. This confers onthe NK-92 cells the capability of likewise specifically targeting thecancer cells present in the individual host. This has the effect ofenhancing the specificity of the cytotoxic activity of the NK-92 cellstoward the cancer cells of that individual. The corresponding processwold be carried out for each host suffering from cancer, takingadvantage of the idiosyncratic specificity of the CTL targeting moietyin each case.

[0063] Methods of treating. The natural killer cells of the inventionare employed in methods of treating biological samples in order to purgethem of cells from a cancer, a malignancy, or a tumor, or cells infectedby a pathogenic virus. The NK cells include by way of nonlimitingexample, NK-92, and modified NK-92 cells, such as NK-92MI and NK-92CI,as well as other modified NK-92 cells envisioned within the scope ofthis invention. The NK-92MI and NK-92CI cells are modified bytransfection with vectors that result in the secretion of IL-2. Inaddition, any of the NK-92, NK-92MI, and NK-92CI cells may be treatedsuch that they maintain the cytolytic activity of the untreated cellsbut cannot proliferate. The NK cells so treated may also be equivalentcell lines which have the properties such as cytotoxicity andNK-specific cell surface markers described herein. Malignancies of theimmune system, the lymphatic system, and the hematopoietic system may betreated by the methods of the invention. In addition, formed tumors andsolid tumors may also be treated. Infections by pathogenic viruses, suchas HIV, EBV, CMV, and herpes may also be treated.

[0064] Treating a biological sample. In vitro biological samples may betreated experimentally or therapeutically in order to eliminatemalignant cells, or virus-infected cells, in an effective manner. Thesample may be drawn from a mammal and maintained in vitro in anappropriate culture medium. Such media are well known to workers ofskill in cell biology, cellular immunology, and oncology. Media and cellculture techniques are presented in general terms in, for example,Freshney, R. I., “Culture of Animal Cells, 3rd Ed.”, Wiley-Liss, NewYork (1994), and in Martin, B. M., “Tissue Culture Techniques, AnIntroduction”, Birkhauser, Boston, Mass. (1994), which are incorporatedherein by reference. The biological sample is established in culture invitro, and contacted with a medium that includes the natural killercells of the present invention. The cytolytic activity of the NK cellseffectively eliminates the malignant cells or the virus-infected cellsfrom the sample. The prevalence and depletion of the target cells may betraced by any of a number of methods well known to those of skill in thefields of cell biology and cellular immunology. These include indirectimmunofluorescence microscopy to assay for intact tumor cells orvirus-bearing cells, fluorescent-activated cell sorting, chromiumrelease assays, and the like.

[0065] Treating a cancer or virus infection ex vivo: purging. Thepresent invention additionally encompasses the ex vivo treatment of abiological sample suspected of containing cancer cells or virus-infectedcells by contacting the sample with the NK cells of the invention. Thebiological sample is drawn from the body of a mammal, such as a human,and may be blood, bone marrow cells, or similar tissues or cells from anorgan afflicted with a cancer. Methods for obtaining such samples arewell known to workers in the fields of cellular immunology, oncology,and surgery. They include sampling blood in well known ways, orobtaining biopsies from the bone marrow or other tissue or organ. Thecancer cells or virus-infected cells contained in the sample areeffectively eliminated due to the cytotoxic activity of the NK-92 cells.The sample may then be returned to the body of the mammal from which itwas obtained.

[0066] The NK-92 cells used to treat the sample may be freely suspendedin the medium. It is generally preferred that the purged sample, priorto being returned to the body of the mammal from which it was obtained,be rid of NK-92 cells that may continue growing, since they aroseoriginally from a proliferating lymphoma. The invention envisionsseveral ways of accomplishing this objective. In one embodiment, the NKcells, prior to use, are irradiated with γ rays or with ultravioletlight to the extent that they maintain their cytolytic activity but arenot capable of growth. In an additional embodiment, the NK cells arepermanently immobilized on a macroscopic solid support. The support withthe NK cells attached may then be physically separated from the cells ofthe biological sample, for example by centrifugation, or filtration witha column which permits the unbound cells of the sample to pass through,or like technique. Suitable solid supports include particles ofpolyacrylamide, agarose, cellulose, Sepharose™ (Pharmacia, Piscataway,N.J.), celite, and the like, and may be supplied with groups such as analdehyde, carbonyldiimidazole, broamoacetyl, epichlorhydrin, and thelike, which are activated for reaction with cell surface groups. Theactivated groups on the support react with groups such as amino orcarboxyl groups, for example, on the cell surface, thereby immobilizingthe cells on the support.

[0067] In yet a further embodiment, the NK cells may be modified with avector directing the synthesis of a cellular component sensitive to anagent, such that when the agent is administered to the ex vivo sample,the NK-92 cells are inactivated. Examples of such agents includeacyclovir or gancyclovir, by way of nonlimiting example. Functionallyequivalent vectors, directing the synthesis of alternative cellularcomponents sensitive to different agents, are also envisioned within thescope of this embodiment.

[0068] The NK cells to be used in the methods of the invention mayrequire a cytokine such as IL-2 to maintain their functionaleffectiveness as cytolytic cells. The cytokine may simply be added tothe ex vivo preparation. Alternatively, if desired, a modified NK-92cell bearing a vector directing the constitutive synthesis of thecytokine may be employed. In this way the necessity of furnishingexogenous cytokine is avoided.

[0069] Treating a cancer or virus infection in vivo: administeringNK-92. A further method of the invention is directed toward treatment ofa cancer or a virus infection in vivo in a mammal using NK-92 cells. Thecells are administered in a variety of ways. By way of nonlimitingexample, the cells may be delivered intravenously, or into a body cavityadjacent to the location of a solid tumor, such as the intraperitonealcavity, or injected directly within or adjacent to a solid tumor.Intravenous administration, for example, is advantageous in thetreatment of leukemias, lymphomas, and comparable malignancies of thelymphatic system, as well as in the treatment of viral infections.

[0070] As has been described in detail in the preceding section, it isdesirable to employ methods that eliminate or ablate the NK-92 cellsafter they have effectively lysed (or otherwise destroyed) the targetcells. Certain methods described above may be employed for this purpose,namely, use of irradiated NK-92 cells, and use of NK-92 cells harboringa vector directing the synthesis of a cellular component sensitive to anagent, such that when the agent is administered, the NK-92 cells areinactivated, and equivalent methods. When the cells produce such acomponent sensitive to the specific agent, administration of the agentto the mammal is effective to inactivate the NK-92 cells within themammal.

[0071] The NK-92 cells may be administered in conjunction with acytokine such as IL-2 in order to maintain the functional effectivenessof the cells as cytotoxic effectors. As used to describe the invention,the term “in conjunction” indicates that the cytokine may beadministered shortly prior to administration of the NK-92 cells, or itmay be given simultaneously with the cells, or shortly after the cellshave been administered. The cytokine may also be given at two suchtimes, or at all three times with respect to the time of administeringthe NK-92 cells. Alternatively, NK-92 cells harboring a vector directingthe constitutive synthesis of the cytokine may be employed in the invivo method of treating a cancer. This effectively eliminates the needto furnish exogenous cytokine.

[0072] The following examples are included to illustrate the inventionand not to limit the invention. All publications or references cited inthe present specification are hereby incorporated by reference. Alldeposits referred to in the present specification are in the process ofbeing submitted to ATCC.

EXAMPLES Example 1

[0073] NK-92 Cells. NK-92 cells (Gong et al. (1994)) were derived fromcells obtained from a patient suffering from non-Hodgkin's lymphoma.PBMC from the patient were cultured in enriched alpha MEM supplementedwith fetal calf serum (12.5%) and horse serum (12.5%) plus 10⁻⁶ Mhydrocortisone and 1000 U/mL of recombinant human IL-2 (rhIL-2). Cellswere cultured at 37° C. in humidified air containing 5% CO₂. Subcultureswere made after 4 weeks, and propagated indefinitely with twice-weeklychanges in medium. In these later stages the hydrocortisone could beomitted without any effect on cell growth. This culture has beendesignated NK-92 and has been deposited with the American Type CultureCollection (ATCC; Rockville, Md.) under designation CLR-2407.

[0074] The cells have the morphology of large granular lymphocytes. Thecells bear the CD56^(bright), CD2, CD7, CD11a, CD28, CD45, and CD54surface markers. In contrast, they do not display the CD1, CD3, CD4,CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, and CD34 markers. Growthof NK-92 cells in culture is dependent upon the presence of recombinantinterleukin 2 (IL-2), with a dose as low as 10 IU/mL being sufficient tomaintain proliferation. IL-7 and IL-12 do not support long-term growth,nor do other cytokines tested, IL-1α, IL-6, tumor necrosis factor α,interferon α, and interferon γ.

Example 2

[0075] Cytotoxic Activity of NK-92 against Different Leukemic CellLines. The cytotoxic activity of NK-92 against K562, Daudi, TF-1,AML-193, and SR-91 cells was determined (Gong et al. (1994)). K562(erythroleukemia) and Daudi (Burkitt) lymphoma cell lines were obtainedfrom ATCC. They were maintained in continuous suspension culture in RPMI1640 medium supplemented with 10% fetal calf serum (FCS). TF-1 is amyelomonocytic cell line (Kitamura et al., J. Cell Physiol. 140:323-334(1989)) that requires the presence of medium containing 2 ng/mL of humanGM-CSF. AML-193 is a myeloid cell line that is maintained in thepresence of 10% 5637-conditioned medium (Lange et al., Blood 70:192-199(1987)). Both TF-1 and AML-193 cells were obtained from Dr. D. Hogge,Terry Fox Laboratory, University of British Columbia, Vancouver, BC.SR-91 is a cell line with features of early progenitor cells establishedby Gong et al. (1994) from a patient with acute lymphoblastic leukemia(ALL) (Klingemann et al., Leuk. Lymphoma, 12, 463-470 (1994). It isresistant to both NK and activated-NK (A-NK) cell cytotoxicity. SR-91 isalso maintained in RPMI 1640/10% FCS. This cell line can be renderedsensitive to killing by NK-92 by treatment with cytokine. Naki et al.,“Induction of sensitivity to the NK-mediated cytotoxicity by TNF-αtreatment: Possible role of ICAH-3 and CD44,” Leukemia, in press.

[0076] The cytotoxic activity of NK-92 (effector) against these targetcells was assessed by means of a ⁵¹Cr release assay (Gong et al. (1994))using the procedure described by Klingemann et al. (Cancer Immunol.Immunother. 33:395-397 (1991)). The percentage of specific cytotoxicityin triplicate specimens was calculated as:

% ⁵¹Cr release=(average experimental cpm−average spontaneouscpm)×100/(average maximum cpm−average spontaneous cpm).

[0077]FIG. 1 presents the results of this determination. It is seen thatNK-92 cells kill K562 and Daudi cells with high efficiency. Even at thelow E:T ratio of 1:1, 83% of K562 cells and 76% of Daudi cells werekilled by NK-92 cells. Susceptibility to killing by NK-92 cells waslower for TF-1 cells (23% at E:T=1:1) and for AML-193 cells (6% atE:T=1:1). SR-91 cells appear to be resistant to the cytotoxic effect ofthe NK-92 cells. Without wishing to be bound by theory, it is believedthat SR-91 cells lack adhesion molecules necessary to mediate initialbinding with NK-92 cells.

Example 3

[0078] Cytotoxicity of NK-92 against Leukemia, Lymphoma, and MyelomaTarget Cell Lines. K562 (Ph-chromosome positive [Ph⁺] erythroleukemia),HL60 (promyelocytic), U937 (myelomonocytic), KG1a (variant subline ofthe AML cell line KG1), DHL-10 (B-cell lymphoma), Daudi (Burkitt'slymphoma), Raji (B-cell lymphoma), Jurkat (T-cell lymphoma), U266 (IgEmyeloma), NCI H929 (IgA myeloma), and RPMI 8226 (myeloma, light chainsecreting) cell lines were obtained from ATCC. The lymphoma-derived celllines Ly3 (B-lineage, diffuse large cell), Ly8 (immunoblastic), andLyl3.2 (T-lineage, diffuse large cell) were provided by Dr. H. Messner,Toronto, Ontario. Their characteristics have been described (Chang etal., Leuk. Lymphoma 19:165 (1995)). All lines were maintained in RPM11640 medium supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 0.1mM nonessential amino acids, 50 U/mL penicillin, 25 mM HEPES (StemCellTechnologies), and 5% heat-inactive FCS (RPMI/5% FCS) at 37° C. in ahumidified atmosphere of 5% CO₂ in air.

[0079] Cell lysis was determined by a 4-hour ⁵¹Cr release assay usingvarious E:T ratios. To allow for comparison, PBMCs from normal donorswere activated with IL-2 (500 U/mL) for 4 days and ⁵¹Cr release measuredagainst the same target cells concurrently. The mean of two separateexperiments is presented.

[0080] Results of NK-92-mediated cytotoxicity (⁵¹Cr release assay)against various leukemia, lymphoma, and myeloma target cell lines aresummarized in Table 1. For comparison, lysis of the same tumor targetcells was also tested in the same experiment with PBMCs obtained fromnormal donors. Those cells had been activated by IL-2 (500 U/mL) for 4days prior to testing. Results show that NK-92 cells very effectivelylyse all target cells tested. High cytotoxicity is observed even at thelow E:T ratio of 1:1. The cytotoxicity achieved with these cells issignificantly higher than that observed with normal (allogeneic) PBMCsactivated under optimal conditions with IL-2 for all the target cellsexcept RPMI 8226 and U266. TABLE 1 Cytotoxic activity of NK-92 cellsagainst various leukemia, lymphoma, and myeloma cell lines Target 50:120:1 10:1 5:1 1:1 HL-60 NK-92 97 90 77 46 40 PBMCs + IL-2 31 26 17 2 0K562 NK-92 68 68 64 59 50 PBMCs + IL-2 63 73 67 51 19 KG1a NK-92 90 9180 67 39 PBMCs + IL-2 15 11 12 6 0 U937 NK-92 99 98 96 91 85 PBMCs +IL-2 57 43 23 13 2 DHL-10 NK-92 95 95 92 94 80 PBMCs + IL-2 60 40 24 195 Daudi NK-92 94 87 71 48 39 PBMCs + IL-2 65 57 29 16 6 Jurkat NK-92 100100 98 93 80 PBMCs + IL-2 67 50 36 27 4 Ly 3 NK-92 63 59 53 42 28PBMCs + IL-2 47 35 18 6 095 Ly 8 NK-92 95 104 102 88 42 PBMCs + IL-2 6765 62 59 44 Ly 13.2 NK-92 104 105 100 97 67 PBMCs + IL-2 61 63 52 4 13Raji NK-92 81 75 74 70 54 PBMCs + IL-2 32 67 57 35 13 NCI H929 NK-92 9489 89 86 51 PBMCs + IL-2 75 58 39 24 5 RPMI NK-92 82 72 70 72 41 8224PBMCs + IL-2 95 83 81 67 25 U266 NK-92 84 77 85 81 53 PBMCs + IL-2 84 7473 56 21

Example 4

[0081] Effect of Deprivation of IL-2 on Cytotoxic Activity of NK-92. Totest how long NK-92 cells would maintain their cytolytic activitywithout IL-2 present in the culture medium, NK-92 cells were deprived ofIL-2 and ⁵¹Cr-release was measured in 24-hour intervals. Results,summarized in FIG. 2, suggest that the cells maintain full cytotoxicactivity for at least 48 hours. Thereafter, the activity dropsprecipitously to negligible levels. Thus, for short-term purging, IL-2does not have to be present in the cultures to achieve a suitableeffect.

Example 5

[0082] Co-culture of K562-neo′ Cells with PBMC'S and NK-92. Thetransfection of the K562 cells with the neomycin-resistance (neo′) genehas been described (Wong et al., Bone Marrow Transplant 18:63 (1966)).Briefly, 5×10⁷ K562 cells were suspended in 0.8 mL RPMI 1640/5% FCS andincubated on ice for 10 minutes with 30 μg of the pMC1-Neo plasmid(provided by Dr. K. Humphries, Terry Fox Laboratory, Vancouver, BC). Thecells were then exposed to a single voltage pulse (125 μF/0.4kV) at roomtemperature, allowed to remain in buffer for 10 minutes, transferredinto 25-cm² tissue culture flasks (Falcon, Lincoln Park, N.J.), andincubated at 37° C. in a humidified atmosphere of 5% CO₂ in air for 2days. Transfected cells were selected in 0.8% Iscove's methylcellulosemedium (StemCell Technologies) supplemented with 30% FCS, 10⁻⁴ M2-mercaptoethanol, and 2 mM glutamine, containing 0.8 mg/mL G418(neomycin) (Gibco-BRL, Grand Island, NY). Neo′ clones of K562 cells wereidentified after 2 weeks, plucked, and maintained in RPMI/10% FCScontaining 0.8 mg/mL neomycin. K562-neo′ cells cultured for 2 daysshowed a neo′ clonogenic cell doubling time of 36-42 hours.

[0083] Normal PBMCs (10⁴/mL) were spiked with 10% K562-neo′ cells, andNK-92 cells were added to yield different effector:target (E:T) ratiosof NK-92:K562-neo′ cells. (Wong et al. (1996)). Briefly, PBMCs weresuspended in enriched alpha medium (Myelocult™) as described above. Thismedium has been shown to provide optimal conditions for supporting bothIL-2 activation of PBMCs and hematopoietic progenitor cell function(Klingemann et al., Exp. Hematol. 21:1263 (1993)). The finalconcentration of PBMCs in 35-mm tissue culture dishes (Corning, EastBrunswick, N.J.) was 1×10⁶/mL, and the proportion of input K562-neo′cells was kept at 10% for all experiments. Various numbers of irradiated(1000 cGy) NK-92 cells (see Examples 7 and 8) were added, resulting invarious E:T ratios as specified in Table 2. These mixtures were culturedin an atmosphere of 5% CO₂ in air for 4 or 48 hours at 37° C. with andwithout IL-2 (500 units/mL).

[0084] After the culture, cells were washed in RPMI/5% FCS, 10³ cellswere suspended in 0.8% Iscove's methylcellulose containing 0.8 mg/mLneomycin, and 1.1-mL volumes were plated in 3-mm petri dishes. After 7days at 37° C. in a humidified atmosphere of 5% CO₂ in air, colonieswere counted. The number of neo′ colonies provided a measure for thenumber of surviving clonogenic K562-neo′ cells present in the cellsuspension originally plated. Percent survival values for co-culturescontaining various numbers of NK-92 cells were determined by comparingthe number of clonogenic K562-neo′ cells present in test co-cultureswith the number of those present in control co-cultures (no NK-92 cellsadded) and harvesting after the same period of incubation. At an inputnumber of 10³ K562-neo′ cells prior to purging, the absolute number ofclonogenic K562-neo′ cells after 4 hours with no NK-92 cells present was6400±820 cells and after 48 hours 28,300±2100 cells. The mean ± SEM offour to eight experiments is reported.

[0085] When only PBMCs were plated in the neomycin-containingmethylcellulose medium, no colonies were ever observed. To quantitatethe purging capacity of NK-92 cells, PBMCs were spiked with 10%K562-neo′ cells and cultured for 4 or 48 hours in medium in the presenceor absence of IL-2. Results, summarized in Table 2, show that NK-92cells used at E:T ratios of 10:1 and 5:1 eliminated the K562-neo′ cellsfrom PBMCs, and that very low survival was observed at E:T of 1:1. Thepresence of IL-2 during the purging did not result in any increase inthe number of K562 cells purged compared to no IL-2 (Table 2). TABLE 2Purging effect of NK-92 cells NK-92:K562- % Survival (−IL-2) % Survival(+IL-2) neo′ of K562-neo′ of K562-neo′ E:T Ratio 4 hrs. 48 hrs. 4 hrs.48 hrs. 10:1  0 0 0 0 5:1 0 0 0 0 1:1 10.5 ± 2.1  15.4 ± 7.2  6.5 ± 3 15.2 ± 5.9  0.1:1     56 ± 14.1 68.5 ± 19.5 54.4 ± 13  69.6 ± 16.8

Example 6

[0086] Effect of NK-92 Cells on Hematopoietic Progenitor Cells. Theeffect of NK-92 cells on PBMCs was determined (Cashman et al., Blood75:96 (1990)). Briefly, normal PBMCs were co-cultured with irradiated(1000 cGy) NK-92 cells (see Examples 7 and 8) for 2 days. Cells werethen plated in replicate 1.1-mL aliquots of methylcellulose-containingmedia at densities adjusted to give approximately 10-100 large coloniesof erythroid cells (from burst-forming units-erythroid [BFU-E]),granulocytes and macrophages (from colony-formingunits-granulocytelmacrophage [CFU-GM]), and combinations of all of these(from CFU granulocyte/erythroid/macrophagelmegakaryocyte [CFU-GEMM]).Colonies were counted under an inverted microscope 2 weeks later.

[0087] The number and growth kinetics of clonogenic hematopoietic cellswere quantified at a 1:1 ratio of NK-92:PBMC after 2 days of co-culturewith irradiated (1000 cGy) NK-92 cells. The cells were plated instandard methylcellulose and counted 2 weeks later. Results obtainedfrom three different normal donors are presented as percentage of normalcontrols in Table 3. No growth inhibitory effect on hematopoieticprogenitors by NK-92 cells was noted. TABLE 3 Effect of NK-92 cells oncolony formation of normal hematopoietic progenitor cells. ExperimentNumber CFU-GEMM BFU-E CFU-C 1 100 46 94 2 200 98 64 3 33 104 103

Example 7

[0088] γ-Irradiation of NK-92 Cells. NK-92 cells were irradiated in T25flasks (Corning, Newark, N.J.) with the dose indicated using a cesiumsource (Cis-US, Bedford, Mass.). A dose range of 200-2000 cGy wastested. After irradiation, the cells were washed twice in RPMI,resuspended in medium, and cultured for 72 hours at 37° C. in thepresence of 500 IU/mL IL-2. Cytotoxicity (⁵¹Cr-release assay) wasperformed with these cells as described above in Example 2. Prior toperforming the ⁵¹Cr-release assay, the cells were left for 24 hours inmedium supplemented with IL-2 to allow for recovery. Proliferation wasassessed by means of a ³H-thymidine incorporation assay. Prior to adding³H-thymidine (0.5 μCi/cell), NK-92 cells were resuspended inthymidine-free RPMI. Uptake of ³H-thymidine was measured in a liquidscintillation counter 4 hours later. (Klingemann et al., Leuk. Lymphoma12:463 (1994)). The counts per minute (cpm) from three differentexperiments are presented.

[0089] Clinical use of this cell line to purge cancerous cells requiresthat NK-92 cells not undergo significant growth and proliferation. Thiswas achieved by irradiating the cells. Proliferation, as measured by ³Hincorporation, was effectively reduced at a dose of 1000 cGy (Table 4).The cytotoxicity of NK-92 cells after administration of variousradiation doses is presented in FIG. 3. At doses up to 1000 cGy anessentially undiminished cytolytic response was maintained. TABLE 4Effect of irradiation on the proliferation of NK-92 cells ExperimentRadiation dose (cGy) Number 0 500 1000 1500 2000 1 5766 3071 406 125 1142 4236 2411 1216 1192 562 3 3994 2046 824 689 748

Example 8

[0090] Radiation susceptibility of NK-92 cells. NK-92 cells wereirradiated by a γ ray source (Gammacell 40, Atomic Energy of Canada.Ltd., Canada). A dose range of 100-3000 cGy was tested. Afterirradiation, the cells were washed and resuspended in culture mediumwith rhIL-2. Colony assays, viability and cytotoxic activity of theirradiated NK-92 cells were performed using standard techniques (Yan etal., Leukemia, 7:131-139 (1993)). To quantify clonogenic NK-92 cells,NK-92 cells (500 cells per mL culture medium) were cultured in a 0.3%agar-based medium supplemented with 12.5% FCS, 12.5% horse serum, 2 mML-glutamine, 100 μg/mL penicillin 50 μg/mL streptomycin, 10⁻⁵ Mmercaptoethanol, and 500 U/mL rhIL-2 at 37° C. for 14 days. Anadditional aliquot of 500 U/mL rhIL-2 was added at day 7 during theculture. Triplicate cultures were performed for each data point.

[0091] The viability of NK-92 cells, determined by trypan blue staining,and the recovery of the ability of the NK-92 cells to generate coloniesafter exposure to various radiation doses is shown in FIGS. 4 and 5,respectively. The NK-92 cells maintained substantial survival for 3 or 4days after exposure to high doses of radiation (1000-3000 cGy). However,in vitro clonogenic NK-92 cells were significantly depleted after lowdoses of radiation and totally eliminated by doses above 300 cGy. FIG. 6shows the cytotoxicity of NK-92 cells to K562, HL60 and 2patient-derived leukemic samples after exposure to the different dosesof radiation. Doses of 300, 500, and 1000 cGy allow for substantialcytolysis against leukemic cell lines and primary leukemias 1-2 daysafter radiation.

[0092] These experiments suggest that NK-92 cells, irradiated to anextent that renders them nonclonogenic, retain their cytolytic activityagainst a wide spectrum of target cells. They therefore may be used exvivo in the purging of tumor cells as well as in the treatment ofvarious cancers in vivo.

Example 9

[0093] Cytolysis of human primary leukemic cells by NK-92.

[0094] a. Patient-derived leukemic samples. Samples were obtained, withinformed consent, during routine diagnostic blood studies or bone marrow(BM) aspirates from patients with newly diagnosed or relapsed leukemias.9 acute myeloid leukemia (AML) cases, 11 chronic myeloid leukemia (CML)cases (6 chronic phase, 1 accelerated phase and 4 blast crisis), 14B-lineage-acute lymphoblastic leukemia (ALL) cases (13 pre-B-ALLs and 1B-ALL) and 6 T-ALL cases, were studied (see Table 5). Blast-enrichedmononuclear cells were isolated by Ficoll Hypaque (Pharmacia,Piscataway, N.J.) density gradient separation and washed in RPMI 1640medium.

[0095] b. Effector cells. NK-92 cells were cultured and maintained inα-MEM medium supplemented with 12.5% FCS, 12.5% horse serum and rhIL-2(500 U/mL Chiron, Emeryville, Calif.). TALL-104 cells (aMHC-unrestricted human cytotoxic T cell clone, generously provided byDrs. D. Santoli and A. Cesano, The Wistar Institute, Philadelphia) weremaintained in Iscove's modified Dulbecco's medium supplemented with 10%FCS and rhIL-2 (100 U/mL) (Cesano et al., Blood, 87:393-403 (1996)).Another human NK cell clone, YT, was maintained in RPMI 1640 medium with10% FCS and rhIL-2 (100 U/mL) (Yodoi et al., J. Immunol., 134:1623-1630(1985)).

[0096] c. Cytotoxicity assays. The cytotoxic activity of non-irradiatedNK-92 and responding T cells against leukemic targets was measured in astandard 4-hour chromium release assay (CRA). Some of the samples werealso measured in an 18-hour CRA. A fixed number of ⁵¹Cr-labeled targetcells (5×10³/well) was tested for susceptibility to 4 effector cellconcentrations. ⁵¹Cr release of target cells alone (spontaneous release,determined by placing target cells in 5% Triton) was always <25% ofmaximal ⁵¹Cr release. CRA data were expressed as specific lysis (%) at agiven effector:target (E:T) ratio or were converted to lytic units (LU)defined as the number of effectors resulting in 30% lysis of targetcells (Cesano et al., Cancer Immunol. Immunother., 40:139-151 (1995)).The degree of sensitivity of patient-derived leukemic cell targets toeach effector was defined as insensitive (−/+: <10/10-19% lysis),sensitive (++/+++/++++: 20-29/30-39/40-49% lysis) and highly sensitive(+++++/++++++: 50-59/>60% lysis) at an E:T ratio of 9:1.

[0097] d. Results: Cytolysis of human primary leukemic cells by NK-92cells. The sensitivity of patient-derived leukemic cells to thecytotoxic effect of NK-92 cells is summarized in column 4 of Table 5. Ofthe 40 patient-derived leukemic samples shown in Table 5, 26 (65%) weresensitive or highly sensitive to NK-92 mediated in vitro cytotoxicity.Six of the samples that were insensitive to the NK-92 cells in thestandard 4 hr CRA (sole or first entries), became sensitive after 18hours incubation (second entries, enclosed in parentheses). Leukemiablasts derived from 6 out of 9 (67%) AML, 6 of 6 (100%) T-ALL and 6 of14 (43%) B-lineage-ALL were either sensitive or highly sensitive to theNK-92 mediated lysis. 7 of 8 acute leukemia samples which demonstratedhigh sensitivity to the cytotoxic effect of NK-92 cells were derivedfrom relapsed patients and 1 was from a newly diagnosed patient. Out of11 CML samples, 8 (73%) were sensitive (5 in chronic phase) or highlysensitive (2 in blast crisis; 1 in accelerated phase) to the NK-92mediated cytolysis (Table 5).

[0098] In comparison, the last two columns in Table 5 present resultsobtained with cell lines known in the field to have cytolytic activityagainst tumor cells, namely, TALL-104 cells and YT cells. Only 16 out ofthe 37 leukemic samples tested (43%) were sensitive (4 AMLs, 5 B-lineageALLs and 3 CMLs) or highly sensitive (1 AML, 1 B-lineage-ALL and 2 CMLs)to the MHC unrestricted cytotoxic T cell clone TALL-104 mediatedcytolysis. Leukemias sensitive to the TALL-104 cells were notconsistently sensitive to NK-92 cells, and cells that were lysed byNK-92 cells were not always lysed by TALL-104 cells. In addition, thecytolytic activity of TALL-104 cells was usually detected only after 18hours of incubation (second entries, enclosed in parentheses). Only fourof 16 (25%) of the target samples that were lysed at 18 hours were alsolysed in the standard 4 hr CRA. The remaining 12 (75%) responded onlyafter the 18 hour incubation, with the response being generally lowerthan that observed with the NK-92 cells of the invention. Withoutwishing to be bound by theory, these observations may be due to thepossibility that 1) different target structures are recognized byTALL-104 vs NK-92 cells, or 2) a different pathway may be involved inthe NK-92 and in the TALL-104 cell mediated cytolysis.

[0099] The majority of leukemic samples treated with YT cells, the otherNK-like clone tested, were found to be resistant, with the exception of2 samples (a CML in blast crisis and a T-ALL) (see Table 5).

[0100] In conclusion, the NK-92 cells of the invention are surprisinglyand significantly more effective in lysing patient-derived tumor cells,and exert their effect in a shorter time, than do the cells from twocytolytic cell lines known in the field. TABLE 5 Cytotoxicity of NK-92,T-ALL104 and YT Clone to Patient-Derived Leukemic Cells^(a) DiseaseBlast (%) in Cytotoxic Sensitivity Patient Status Sample NK-92 TALL-104YT AML  1 M4- Relapse PB (66%) ++++++ +++++ −  □  2 (M1) Relapse PB(50%) +++++ − −  3 (M3) Relapse PB (50%) +++ (++++) + (++++) − (−)  4(M4) Refractory PB (90%) ++ (++) − (+) − (−)  5 (M2) New BM (90%) +++(+++) + (+++) ND  6 (M4) New BM (97%) − − −  7 (M4) New PB (39%) − (−) −(++) − (−)  8 (M3) New PB (55%) − (++) − (+++) + (−)  9 (M3) New BM(32%) − − − T-ALL  1 Relapse BM (98%) ++++++ − −  2 Relapse PB (85%)++++++ − (−) +++ (+++)  3 Relapse PB (77%) ++++++ − (+) − (−)  4 RelapsePB (60%) +++++ − (−) + (−)  5 New BM (40%) +++ − −  6 New BM (66%) +++ −− B-Lineage-All  1 Relapse BM (78%) +++++ ++++ −  2 New BM (30%) ++++ND ND  3 Relapse BM (75%) +++ (++++) + (++++) ++ (++)  4 New BM (97%) ++(+++) + (+++) − (−)  5 Relapse BM (60%) + (+) − (+) − (−)  6 Relapse BM(80%) − ND ND  7 Relapse PB (80%) − − (−) −  8 New BM (68%) − − −  9 NewBM (33%) − − (+) − 10 Relapse BM (87%) − − (++) − 11 Relapse BM (75%) −(+++) − (++++) − 12 New BM (30%) − − ND 13 New PB 90%) − (+++) − (+++)ND 14 New BM (81%) − − ND CML  1 BC PB (45%) ++++++ +++++ +++  2 AC PB(22%) ++++++ ++ −  3 BC PB (93%) +++++ + −  4 CP PB (15%)D ++++ + −  5CP PB (8%)D ++ (++++) ND ND  6 CP BM (12%)D + (+++) + (+) ND  7 CP BM(10%)D + (+++) + (++++) ND  8 BC PB (60%) + − −  9 BC BM (48%) + − (−) −10 CP PB (21%)D + (++) − (++++) − (−) 11 CP PB (11%)D − − (+++++) − (−)

Example 10

[0101] Cytotoxicity of NK-92 towards human leukemic cell lines. Thefollowing human leukemic cell lines were cultured at 37° C. in 5% CO₂ inRPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum(FCS), L-glutamine and antibiotics: K562 (Chronic myeloid leukemia inblast crisis), HL60 (acute promyelocytic leukemia), KG1(erythroleukemia), NALM6 (acute pre-B lymphoblastic leukemia), Raji(Burkitt's lymphoma), CEM/S (acute T lymphoblastic leukemic cell linesensitive to methotrexate (MTX), a commonly used antitumor drug) as wellas GEM/T (methotrexate-resistant subline of CEM/S) (Mini, E. et al.,Cancer Res. 45:325-330 (1985)).

[0102] NK-92 cells were highly cytotoxic to all the 8 leukemic celllines tested in a 4 hr standard CRA (Table 6). The MTX-sensitive T-ALLcell line CEM/S as well as its MTX-transport resistant subline CEM/Tdisplayed a similar sensitivity to the NK-92 cells. This suggests thattumors that are not responsive to MTX treatment could be treated byadministering NK-92 cells of the instant invention. Table 6 surprisinglyshows highly effective cytolytic activity for NK-92 against all thetarget cells tested. The results obtained at the low E:T ratio of 1:1are especially noteworthy.

[0103] In contrast, the cytolytic cell lines TALL-104 and YT have littleor virtually no cytolytic activity against many of these target cellsunder these conditions; when active, their activity is generally lowerthan that for NK-92 at E:T of 1:1. TALL-104 was cytotoxic to K562, NALM6and HL60 cells, however, Raji cells exhibited only 22.2% lysis at 9:1E:T ratio and KG1 cells, CEM/S as well as CEM/T were resistant. The YTclone did not exhibit significant cytotoxic activity. Activity was foundonly against K562 cells and Raji cells, which showed a 32% and 25% lysisat 9:1 E:T ratio, respectively.

[0104] As shown in Table 6, NK-92 cells of the present invention have asignificantly wider range of action and higher activities than the knowncytolytic cell lines TALL-104 and YT. These activities are higher thanany previously reported values in the field of tumor cytotherapy. TABLE6 Specific Lysis of Human Leukemia Cell Lines by Natural Killer CellClonesNK-92, TALL-104, and YT. Specific Lysis (%) NK92 TALL-104 YTEffector:Target Ratio Target 9:1 3:1 1:1 9:1 3:1 1:1 9:1 3:1 1:1 K56294.1 91.2 82.1 88.5 85.2 72.5 34.2 28.2 18.4 HL60 87.9 75.3 79.6 43.016.0 6.9 2.1 1.1 1.5 KG1 64.6 53.8 43.7 2.7 0.5 0 0.1 0 0 NALM- 72.656.8 52.4 67.8 55.6 33.3 1.0 0.5 0 6 Raji 86.0 75.4 70.0 22.2 10.2 0.325.1 18.0 14.2 TALL- 57.3 53.2 44.1 — — — 3.2 1.4 0.9 104 CEM/S 56.648.8 34.7 2.7 1.6 0.9 0.9 0.4 0.3 CEM/T 57.5 42.1 39.1 1.5 0.6 0.3 1.20.1 0.2

Example 11

[0105] Effect of NK-92 cells on normal human bone marrow hematopoieticcells. Heparinized bone marrow collected from normal donors wasseparated by Ficoll Hypaque density gradient isolation to produce themononuclear cells. Enrichment of hematopoietic cells and depletion of Tcells was achieved by soybean lectin agglutination (SLA) of maturemarrow elements and removal of residual T cells by resetting with sheepred blood cells (Reisner et al., Lancet, 2:327-31 (1981)).

[0106] Hematopoietic cell enriched fractions of normal bone marrows from14 normal donors were tested by standard CRA to determine theirsusceptibility to lysis by NK-92 cells. All of the normal bone marrowsamples were insensitive to NK-92 mediated cytolysis (FIG. 7).

Example 12

[0107] In vivo leukemogenesis of NK-92 cells in SCID mice.

[0108] a. Experimental animals. Severe combined immunodeficient (SCID)mice (CB17 scid/scid and pfp/Rag-2) (6 to 8 weeks old; Taconic Farms,Germantown, N.Y.) were maintained in microisolator cages under sterileconditions with a specific pathogen-free environment. To determine thepotential of NK-92 cells to induce leukemia in vivo, 2×10⁷ viable NK-92cells in 0.3 mL phosphate buffered saline (PBS) were administrated byeither intraperitoneal (I.P.) or intravenous (I.V.) route every otherday for 5 injections in each animal. For subcutaneous (S.C.)inoculations, 2×10⁷ NK-92 cells were injected in the right flank of SCIDmouse, as described previously (Yan et al., Blood, 88:3137-3146 (1996)).Thereafter, all the experimental animals were administered rhIL-2, 5×10⁴U every other day for 2 weeks by S.C. injection. Survival of the animalswas followed for at least 6 months after inoculation.

[0109] b. Tissue analysis. From each group, 2 SCID mice were sacrificedat the end of observation and tissues from peripheral blood, bonemarrow, spleen, liver, kidney, lung, and brain were collected forhistopathological and/or fluorescence activated cell sorting (FACS)analysis. Tissue sections from sacrificed SCID mice were fixed in 10%neutral buffered formalin, dehydrated and embedded in paraffin,sectioned and stained according to standard histological techniques.

[0110] Viable cells recovered from various tissues were stained byfluorescein isothiocyanate-conjugated (FITC) or phycoerythrin-conjugated(PE) Mab, as described (Yan et al., (1996)). A FACS scan flow cytometer(Becton Dickinson) was used for analysis. Monoclonal antibodies (Mabs)directed against the respective human cell surface antigens were usedfor determination of their presence: CD2, CD3, CD5, CD7, HLA-DR, CD45,CD56 (Becton Dickinson). A fluorescein isothiocyanate (FITC)-conjugatedrat anti-mouse Mab mCD45 (Boehringer-Mannheim, Indianapolis, Ind.) wasused for characterization of murine leukocyte common antigen.

[0111] c. Leukemogenesis. CB-17 scid/scid mice as well as pfp-Rag-2 micewere inoculated with NK-92 cells by I.V. (n=3, for each group), S.C.(n=2, each group) and I.P. (CB-17: n=8; pfp-Rag-2: n=3) injection.Survival of the animals was followed at least 6 months afterinoculation. At the end of the six month period, all animals appearedhealthy; there was no hepatosplenomegaly, lymphadenopathy or leukemicnodular growth, which would have indicated leukemia development.Leukemic cellular infiltration was not detected in the different tissuesof the sacrificed animals by histopathology. No cells of human originwere detectable in the tissues by FACS analysis.

Example 13

[0112] Comparison of antileukemic effect of NK-92 cells with LAK, NK andT cells against human leukemic cell lines. To isolate the NK cellpopulations, a Ceprate^(R) cell separation system based on avidin-biotinimmunoaffinity (CellPro, Bothel, Wash.) was used to purify a CD56+ cellfraction from cultured LAK cells. Briefly, the harvested cells werewashed and resuspended in PBS with 1% bovine serum albumin (BSA). Toeach 1-2×10⁸ cells/mL, 40 μL primary monoclonal antibody (mouseanti-human CD56) was added and the cells were incubated at 4° C. for 25minutes. After incubation, the cells were washed and resuspended to aconcentration of 1×10⁸ cells/mL in PBS with 1% BSA. Then, to each one mLcell suspension, 20 μL biotin labeled rat antimouse IgG1 antibody wasadded and the cells were incubated again at 4° C. for 25 minutes. Afterincubation, the cells were washed and resuspended at a concentration of1×10⁸ cells per mL in PBS with 5% BSA and slowly passed through theavidin column. The CD56+ cells were captured; other cells, including theT cell fraction, were eliminated from the column. After washing thecolumn, the adherent cells were then disassociated from the column byagitation and elimination. After separation, the NK cell-enrichedpopulations contained >85% CD56+CD3− NK cells. The majority of the othercells in the fraction (>95%) were CD3+CD56− T cells.

[0113] To generate leukemia-reactive allocytotoxic T lymphocytes (CTLs),peripheral blood mononuclear cells (PBMC) isolated from normal donorswere cultured with irradiated leukemic stimulating target cells andirradiated autologous PBMC as feeder cells. Cultures were started in 60well plates at 1000 responder cells per well in RPMI 1640 mediumcontaining 15% human serum and rhIL-2 100 U/mL at 37° C., 5% CO₂. Theratios of stimulator cells and feeder cells to responder cells were 5:1and 10:1, respectively. After 10-12 days culture, CTLs were harvestedfrom growth-positive wells and specific lysis toward leukemic targetcells and K562 cells was quantitated by ⁵¹Cr-release assay. The CTLswere continuously cultured and fed with stimulator and feeder cells inflasks. After 2-3 weeks culture, the monoclonal antibody OKT3 (OrthoBiotech, Raritan, N.J.) was added to the culture for rapid expansion ofthe CTL lines.

[0114] The antileukemic effects of NK-92 cells, human LAK cells, NKcells (CD56+CD3−; CD56+ in FIG. 8), and T cells (CD3+CD56−; CD3+ in FIG.8) were assessed by measuring in vitro cytolytic activity in standardCRA (FIG. 8, Panels A and B), and by measuring inhibition of leukemiccell xenograft growth in vivo (FIG. 8, Panels C and D) when the effectorcells and targets were co-inoculated subcutaneously into SCID mice. Inorder to evaluate the inhibition of growth in vivo, the area of thesubcutaneous growths of leukemic nodules as a measure of their size wasdetermined once a week after inoculation, and survival of the animalswas also followed. NK-92 cells displayed the highest in vitrocytotoxicity against K562 (FIG. 8, Panel A) and HL60 (FIG. 8, Panel B)of the cells tested, with a mean specific lysis of 89% and 78%,respectively. This was superior to the killing mediated by human LAK(52% and 11%, respectively), NK (72% and 28%, respectively) and T cells(12% and 1.2%, respectively).

[0115] Correspondingly, the NK-92 cells demonstrated more effective invivo inhibition of the growth of K562 (FIG. 8, Panel C) and HL60 (FIG.8, Panel D) leukemic cells xenografts than did the human LAK and NKcells. The results shown in FIG. 8 indicate that the NK-92 cells of thepresent invention have cytolytic activity in vitro and tumor-inhibitingactivity in vivo that is superior to those activities manifested by theknown preparations of cytolytic cells normally present in humans. Theseactivities are therefore unexpected by a worker in the field of tumorcytotherapy.

Example 14

[0116] Comparison of antileukemic effect of NK-92 cells with allogeneicleukemic-reactive CTL cells. To examine the in vivo effects of NK-92cells and other effector cells on the growth of human leukemiaxenografts, 5×10⁶ leukemic target cells alone or mixed with 2×10⁷ NK-92or other effector cells (E:T ratio=4:1) were injected S.C. into SCIDmice. The TALL-104 effector cells were irradiated with 3000 cGy beforeinoculation to prevent leukemogenesis in SCID mice. RhIL-2 wasadministered to the mice, as in Example 13. The Log-rank test andWilcoxon test were used for the comparison of the survival of leukemiabearing SCID mice.

[0117] The antileukemia effect of NK-92 cells was evaluated usingallogeneic leukemia-reactive CTL cells (derived from a patient withT-ALL (TA27)). Both NK-92 and CTL cells activated by exposure to TA27displayed a significantly higher specific cytolysis (70% and 58% at 9:1E:T ratio, respectively) than the other effectors (LAK cells: 22%; NKcells (designated CD56+ in FIG. 9): 38%; TALL-104: 8%; and T cells (CD3+in FIG. 9): 1.5% specific lysis) against the TA27 leukemic cells (FIG.9, Panel A). Correspondingly, the subcutaneous growth of TA27 leukemiccells was inhibited by co-injection of either NK-92 cells oranti-TA27-CTL cells (FIG. 9, Panel B). The survival of those animalswhich were co-inoculated with TA27 leukemic cells plus NK-92 or withanti-TA27-CTL cells was significantly prolonged beyond that of theanimals bearing TA27 leukemia alone (NK-92 cells: p=0.001; TA27-CTLcells: p=0.002; see FIG. 10). In contrast the TALL-104 cells did notshow significant in vitro killing against TA27 leukemic cells by CRA(FIG. 9, Panel A). However, moderate inhibition of the leukemic tumorgrowth in vivo (FIG. 9, Panel B), coupled with a statisticallyinsignificant (p>0.05) increase in survival, was observed in the animalsco-inoculated with TA27 leukemic cells and irradiated TALL-104 cells(FIG. 10).

Example 15

[0118] Antileukemia effect of NK-92 cells in human leukemia xenograftSCID mice model. For study of the in vivo tumoricidal capacity of NK-92cells, leukemic cells derived from a T-ALL patient (TA27), an AIMLpatient (MA26), and a pre-B-ALL patient (BA31) were adoptively grown andexpanded in SCID mice by S.C. inoculation. Leukemic cells recovered fromthe leukemic nodules in the mice (first passage) were used in 5 theseexperiments. The SCID mice in each group were inoculated I.P. with 5×10⁶leukemic cells from the first passage in 0.2 mL PBS, and 24 hours later2×10⁷ NK-92 cells in 0.4 mL PBS were administered by I.P. injection. Theanimals received either 1 dose or a series of 5 doses of NK-92 cellswhich were administered on days 1, 3, 5, 7, and 9, with and withoutrhIL-2, as indicated in the Figures.

[0119] All the human leukemias grew aggressively in SCID mice. Leukemiccells derived from a patient (TA27) with T-ALL and a patient (MA26) withAML M4 leukemia were highly sensitive in vitro to the NK-92 cells (73%and 66% specific killing at 9:1 E:T ratio determined by CRA,respectively), whereas cells from a patient with pre-B-ALL (BA31) wereinsensitive to the NK-92 cells (4% specific killing at 9:1 E:T ratioassessed by CRA). FIG. 11 shows that the survival of mice bearing TA27leukemia was significantly prolonged by the administration of NK-92cells. The median survival time (MST) of the animals with no treatmentor rhIL-2 alone was 72 days (n=5) and 63 days (n=5) (p>0.05),respectively. All these animals died of leukemia. Treatment with NK-92cells (alone or with rhIL-2) increased the MST to 102 days (n=6) and 114days (n=6), respectively, for the 1 dose injection schedule (2×10⁷ NK-92cells, day 1). The MST increased to 160 days (n=6) and 129 days (n=6),respectively, with 5 doses NK-92 with or without rhIL-2 injection (FIG.11). Three animals that received 5 doses of NK-92 cell injections withor without rhIL-2 administration survived without any signs of leukemiadevelopment 6 months after inoculation. There was no significantdifference in survival between the mice receiving treatments with orwithout rhIL-2 administration, whether in the group receiving 1 dose ofNK-92 cells (p=0.75), or the in the group receiving 5 doses (p=0.45).Compared to the group receiving 1 dose of NK-92 cells, with or withoutrhIL-2 treatment, survival was significantly extended in animals thatreceived 5 doses of NK-92 cells without rhIL-2 treatment (p=0.009 andp=0.009, respectively).

[0120] In SCID mice inoculated with human pre-B-ALL (BA31) leukemia,with or without rhIL-2 treatment, the MST were 63 days (n=5) and 64 days(n=5), respectively (see FIG. 12). For the animals that received 5 dosesof 2×10⁷ NK-92 cells, with or without rhIL-2 administration, the MST wasincreased to 79 days (n=5) and 76 days (n=5), respectively. Thesesurvival times were not significantly different from those for theanimals that were not treated by NK-92 cells (p>0.05).

[0121] In animals bearing human AML (MA26), MST was 97 days (n=6) (seeFIG. 13). The MST was extended to 173 days among the animals thatreceived 5 doses of 2×10⁷ NK-92 cells (p<0.01)(n=6). Three of the 6animals that received NK-92 cells remained alive 6 months after leukemiainoculation. Two of these appeared healthy without any signs of leukemiadevelopment. One mouse had an enlarged abdomen indicating residualleukemia. The 6 animals that received NK-92 cells plus rhIL-2 treatmentwere all alive 6 months after leukemia inoculation without any signssuggestive of leukemia development.

[0122] The results presented in FIGS. 11-13 show that in vivo treatmentof leukemic tumors can result in enhanced longevity of the subject mice.The extent of the prolongation of life, and of the improvement in thehealth of the animals, is dependent on the particular leukemic tumorinvolved, and ranges from modest or insignificant (FIG. 12) to verydramatic (FIG. 13). Based on these results, it is concluded thattreatment of tumors in vivo by administering NK-92 cells, depending onthe tumor in question, can be surprisingly effective.

Example 16

[0123] Preparation of Modified NK-92 Cell Lines Secreting IL-2. In orderto generate NK-92 cells that constitutively secrete IL-2, two plasmidsencoding human IL-2 were employed.

[0124] a. Methods. DNA Clones: The MG-hIL-2 vector (FIG. 7) wasgenerously provided by Dr. Craig Jordan (formerly of Somatix Corp.,Alameda, Calif.). The pCEP4-LTR-hIL-2 vector (FIG. 8) was created byexcising the Hin DIII-Bam HI fragment from the MFG-hIL-2 vector,containing the 5′ LTR and hIL-2 gene, and inserting it into thecomplementary sites of the pCEP4 episomal vector backbone (InVitrogen,Carlsbad, Calif.).

[0125] Particle-Mediated Gene Transfer: NK cells were transduced byparticle-mediated gene transfer using the Biolistic PDS-1000/He ParticleDelivery System (BioRad Laboratories, Hercules, Calif.). Cells weretransduced according to the manufacturer's instructions. Briefly, 1.0 or1.6 μm gold particles were coated with 5 μg of DNA using calciumchloride spermidine, and ethanol. NK-92 cells were prepared forbombardment by adherence to poly-L-lysine (Sigma, St. Louis, Mo.) coated35 mm tissue culture plates. Cells were bombarded in an evacuatedchamber (vacuum of 20 inches mercury) and DNA-coated particles wereaccelerated by a 1,100 psi helium pulse. Cells were returned to IL-2supplemented Myelocult media immediately following bombardment andallowed to recover for 24 hours prior to transfer to IL-2-free media.Media was changed periodically. Cells were selected for IL-2-independentgrowth. Preliminary experiments showed heat transfer efficiencies of5-15% were obtained under the conditions used.

[0126] PCR and Southern Blot Analysis: The transfection of the NK-92cells was confirmed by polymerase chain reaction (PCR) analysis of DNAisolated from both the parental and transfected NK-92 cell lines for thepresence of genomic and cDNA forms of the human IL-2 gene. DNA wasisolated using DNAzol (Gibco Life Technologies Inc., Burlington, ON).Briefly, cells were lysed in DNAzol and DNA was precipitated withethanol at room temperature. DNA pellets were collected, washed in 95%ethanol and briefly air dried. DNA was resuspended in 8 mM NaOH at 62°C. and the solution was neutralized with HEPES buffer. DNA wasquantitated by absorbance at 260 nm. Primers flanking exon 1 of thehuman IL-2 gene (forward: 5′-CAA CTC CTG TCT TGC ATT GC-3′ and reverse:5′-GCA TCC TGG TGA GTT TGG G-3′, Gibco Lift Technologies Inc.,Burlington, ON) were used to amplify the DNA (30 cycles, 1 min 95° C., 2min 50° C. and 2 min 72° C.). PCR products were resolved on a 2% agarosegel. For Southern blot analysis, DNA was transferred to Hybond+nylonmembrane (Amersham Life Sciences, Arlington Heights, Ill.) by capillarytransfer in 10×SSC (1.5M NaCl, 1.5M NaCitrate) and fixed by UVcross-linking (StrataLinker Stratagene, La Jolla, Calif.). The blot washybridized with a ³²p radiolabeled human IL-2 probe for 8-12 hours,washed and visualized by autoradiography at −70° C. with Kodak X-OmatXAR film.

[0127] Northern Blot Analysis: Cytokine and chemokine gene expressionwas analyzed by Northern blot analysis. RNA was extracted from parentaland transfected NK-92 cell lines using Trizol reagent (Gibco LifeTechnologies Inc., Burlington, ON) according to the manufacturer'sinstructions. Briefly, cells were lysed in Trizol and the lysateextracted with chloroform. The aqueous phase was then precipitated withisopropanol. The RNA pellet was collected, briefly air-dried and thenresuspended in DEPC-treated water (diethyl-pyrocarbonate; Sigma ChemicalCo., St. Louis, Mo.). RNA was quantitated by determining OD_(260nm).Fifteen micrograms of RNA was resolved on a 1% formaldehyde agarose gelin 1% MOPS (3-[N-Morpholino]propanesulfonic acid, Sigma, St. Louis, Mo.)and blotted as described previously for Southern blot analysis. The blotwas hybridize with ³²p radiolabeled probes for human IL-2 and TNF-α.

[0128] DNA probes for Northern and Southern blot analysis wereradiolabeled by random primer extension. DNA probes for human IL-2 andTNF-α were purified by digestion with appropriate restrictionendonucleases and agarose electrophoresis. The DNA was excised from thegel and purified by centrifugation through a Spin-X tube filter (CorningCostar, Cambridge, Mass.), phenol:chloroform extraction and ethanolprecipitation. DNA probe was labeled with α-³²P-dCTP (Sp. Ac. 3000Ci/mmol; ICN, Montreal, PQ).

[0129] Cytokine Determination: IL-2 production by NK-92 cell -lines wasdetermined by ELISA. Aliquots of 1×10⁶ of the parental or transfectedNK-92 cells were cultured in 8 ml of IL-2 free Myelocult media for 1, 2,and 3 days. Supernatants were collected from at −20° C. until allsamples were collected. Samples were thawed and assayed for IL-2 levelsby ELISA according to the manufacturers' instructions (Quantikine; R&DSystems, Minneapolis, Minn.). The ELISA is a horseradishperoxidase/tetramethylbenzidine based colorimetric assay and the ELISAmicrotiter plates were read at 450 nm (with a 540 nm correction) in amicroplate reader (Model EK309, Bio-Tek Instruments Inc., Winooski,Vt.).

[0130] Irradiation of NK-92 Cells: To determine the sensitivity of bothparental and transfected NK-92 cells to irradiation, cells wereirradiated using a Cis Biolnternational 437c cesium source (Cis-US,Bedford, Mass.). Cells were collected, washed and resuspended in mediumand irradiated in 15 or 50 ml conical centrifuge tubes (BectonDickinson, Franklin Lakes, N.J.). Following irradiation, cells werewashed and resuspended in Myelocult with (for parental NK-92) or without(for transfected cells) IL-2. Cells were cultured for 24, 48 and 72hours and assayed for viability by trypan blue exclusion, forproliferation by ³H thymidine incorporation and for cytotoxicity by⁵¹Cr-release assay (as described above).

[0131] b. Plasmid MFG-hIL-2. For NK cells transfected with the MFG-hIL-2vector, 85-95% of cells died after 4-7 days following transfer tounsupplemented media. A small number of cells, however, remained viable.These were assumed to be cells that had been successfully transfected.However, even with these cells, no viable cells were detectable aftertwo to three weeks. This was expected as the MFG-hIL-2 vector constructdid not contain the genetic elements required for replication andmaintenance in eukaryotic cells such as a mammalian origin ofreplication. Therefore, as the transfected cells were maintained inculture and began to replicate, the vector construct would have beenlost from cells and the cells would have reverted to theirIL-2-dependent phenotype. These cultures were nevertheless propagatedfor several weeks. Surprisingly, a small number of viable cells appearedin the cultures after approximately 4-5 weeks following initial transferof the cells to IL-2-free media. These cells were capable ofIL-2-independent growth upon subculturing to fresh media and appeared tobe stably transfected, maintaining their IL-2 independent phenotypeduring prolonged culturing. Since the vector was unable to replicate,the appearance of stably transfected cells suggests that the vector hadintegrated into the genome of a transfected cell. Since this would be avery rare event, these transfected cells probably arose from one or avery small number of cells. IL-2-independent NK-92 cells arising fromtransfection with the MFG-hIL-2 were denoted as NK-92MI.

[0132] c. Plasmid pCEP4-LTR.hIL-2. Initial observations for cellstransfected with the episomal vector pCEP4-LTR.hIL-2 were identical tothose seen with NK-92MI. The majority of the transfected cells diedwithin 4-7 days following transfer to IL-2-free Myelocult media.However, unlike the NK-92MI cells, the remainder of the cells did notlose their IL-2-independent phenotype or vitality and die after theinitial 2-3 week period. Instead, the cells that were initiallyIL-2-independent were immediately capable of long-term IL-2-independentgrowth and survival. This was expected since the pCEP-LTR.hIL-2 vectorcontains elements that enable it to be maintained in eukaryotic cells asan autonomously replicating genetic element. Therefore, any cell thatwas initially transfected should maintain its IL-2-independent phenotypefor an indefinite length of time. Although cells harboring episomalvectors are not stably transferred by strict definition, these cells areunder constant selection pressure in IL-2-free media in favor of cellsmaintaining the vector. Therefore, these cells are capable of long-termculturing. IL-2-independent NK-92 cells arising from transfection withthe pCEP4-LTR.hIL-2 are denoted as NK-92CI.

[0133] To confirm that NK-92MI and NK-92CI have in fact been transfectedwith hIL-2 gene, PCR analysis was performed on the parental andtransfected cell lines. Primers flanking exon 1 of the hIL-2 gene, whichhas 88 base pairs (bp), were used to amplify DNA isolated from NK-92,NK-92MI and NK-92CI to assay for the presence of the genomic and cDNAforms. Agarose gel electrophoresis of the PCR products from the parentalline revealed a single 263 bp fragment corresponding to the sizeexpected for the DNA fragment amplified from the genomic IL-2 gene (FIG.16, Panel A). However, analysis of both the NK-92MI and NK-92CI productsrevealed two bands, the 263 bp fragment corresponding to the genomichIL-2 gene as well as a 175 bp fragment resulting from the amplificationof the hIL-2 cDNA. To confirm the identity of these DNA fragments,Southern blot analysis with a radiolabeled probe specific for hIL-2probe was performed. As seen in FIG. 16, Panel B, both the 263 bpgenomic fragment and the 175 bp cDNA fragment hybridized with the probe.These data indicate that both NK-92MI and NK-92CI had been successfullytransferred and contain the cDNA for hIL-2.

[0134] d. Analysis of Gene Expression. To analyze expression of specificcytokines in the parental and transfected cell lines, they were analyzedby Northern blot analysis. RNA isolated form the NK-92, NK-92MI, andNK-92CI cells was separated by electrophoresis, transferred to a nylonmembrane and hybridized with probes for the cytokines hIL-2 and hTNF-α(see FIG. 17). Northern blot analysis of IL-2 in these cells revealedthat IL-2 RNA was not detectable in the parental cell line (FIG. 17,Panel A, Lane 1). However, hIL-2 was found in RNA from both the NK-92MIand NK-92CI (Lanes 2 and 3, respectively). Two mRNA transcripts wereseen in NK-92MI, a major RNA species of approximately 1.9 kDa and a lessintense transcript at 2.4 kDa. In NK-92CI, a hIL-2 mRNA transcript ofapproximately 1.4 kDa was detected. As well, a very faint band was seenat 2.5 kDa. These data confirm that the transfected cells expressed IL-2while the parental NK-92 cells did not. The significance of the multiplehIL-2 mRNA transcripts in the two transfectants is not clear, althoughit is possibly a consequence of the different vector constructs.Furthermore, in the case of NK-92MI, the integration of the hIL-2 geneinto the genomic DNA may also have affected the RNA size.

[0135] TNF-α expression in the NK cells was also examined using thistechnique (FIG. 17, Panel B). It is seen that all three lines expressedthe gene for this cytokine. A TNF-α probe hybridized to a 1.6 kDa bandin RNA isolated from NK-92, NK-92MI and NK-92CI (FIG. 17, Panel B).These results indicate that although transfection of NK-92 cells withthe IL-2 gene resulted in expression of the IL-2 in the transfectants,this did not influence the expression of another cytokine.

[0136] e. Secretion of hIL-2. After confirming expression of the IL-2gene by Northern blot analysis, cells were assayed for production andsecretion of hIL-2 by ELISA. Aliquots of 10⁶ NK-92, NK-92MI and NK-92CIcells were plated in 8 mL aliquots and cultured in Myelocult in theabsence of IL-2. Supernatants were collected after 24, 48 and 72 hoursfor IL-2 analysis by ELISA. Background levels of IL-2 were detected inthe supernatant of NK-92 cells at all three time points (2-3 pg/mL).Elevated IL-2 levels were detected in both NK-92MI and NK-92CIsupernatants (Table 7). NK-92MI produced much higher levels of IL-2 incomparison to NK-92CI, with levels ranging from 60× higher after 24hours (9.3 pg/mL vs 549.3 pg/mL) to about 80× higher after 48 hours(15.7 pg/mL vs 1,260.3 pg/mL) and 72 hours (27.2 pg/L vs 2,248.3 pg/mL).TABLE 7 Synthesis of Human IL-2 by NK-92, NK-92MI, and NK-92CI IL-2(pg/ml) in Experiment #1 #2 #3 Ave ± S.D. NK-92 Day 1 0 7 1 2.7 ± 3.8Day 2 0 4 1 1.7 ± 2.1 Day 3 0 3 3 2.0 ± 1.7 NK- Day 1 517 568 545 549.3± 34.7  92MI Day 2 977 1462 1342 1260.3 ± 252.6  Day 3 1872 2610 22631148.3 ± 369.2  NK- Day 1 7 13 8 9.3 ± 3.2 92CI Day 2 14 16 17 15.7 ±1.5  Day 3 52 18 13 27.7 ± 21.2

[0137] f. Comparison of cell surface antigens in NK-92. NK-92MI andNK-92CI. To compare the IL-2-independent transfectants with the parentalcells, NK-92MI and NK-92CI were analyzed for CD2, CD3, CD4, CD8, CD10,CE16, CD28, CD56, ICAM-1, ICAM-2, ICAM-3 and LFA-1 expression byfluorescent activated cell sorting (FACS) analysis. The transfectedcells revealed a pattern of expression identical to tat seen on theuntransfected parental cell line with the exception of the IL-2receptor. FACS analysis of CD25 (the IL-2 receptor α-chain) on NK-92cells indicated that the receptor was expressed on the surface of NK-92cells and that its expression is down-regulated in response to IL-2.This confirmed similar findings obtained in earlier work (Gong et al.,1994). Therefore, NK-92 cells in unsupplemented media had relativelyhigh levels of CD25 on their surface while cells in media supplementedwith as low as 100 U/mL had low levels of CD25 cell surface expression.

[0138] CD25 expression in the high IL-2-producing transfectant NK-92MIwas decreased both in unsupplemented media and in media supplementedwith 100 U/mL or 1000 U/mL of IL-2. These results are consistent withthose seen with the parental cells. Since the levels of endogenouslyproduced IL-2 in NK-92MI were high, down-regulation of IL-2 receptorlevels is expected even in the absence of exogenously administered IL-2.

[0139] Culture of NK-92CI in media supplemented with 100 U/mL and 1000U/mL IL-2 resulted in CD25 upregulation and increased cell surfaceexpression. However, the results for NK-92CI in unsupplemented media arenot as clear. Two distinct populations appear, a population expressingvery low CD25 levels, similar to NK-92MI, and a population expressinghigh levels, similar to the NK-92 parental cells. This suggests thatNK-92CI consists of a polyclonal population consisting of high and lowIL-2 expressing cells rather than a uniform population of cellsexpressing an intermediate to low level of IL-2. Therefore, whencultured in IL-2-free media, the cells expressing high levels of IL-2would have low surface levels of CD25 while low IL-2 expressing cellswould have high CD25 levels on their surface.

Example 17

[0140] Cytotoxicity of NK-92 Transfected to Produce IL-2. To evaluatethe cytotoxicity of these transfected cells, a standard 4 hour⁵¹Cr-release assay was performed to compare the toxicity of the parentalcells to NK-92MI and NK-92CI to the standard test target cells K562 andRaji. The cytotoxicity of NK-92MI and NK-92CI was comparable to thatseen with the parent cells (FIG. 18). The transfected cell lines showcytotoxic activities against K562 and Raji that are very similar to thatof the parental cells. Cytotoxicity of NK-92 against K562 ranged from 82to 67% while NK-92MI and NK-92CI had cytotoxicity ranges of 77 to 62%and 82 to 62%, respectively. For Raji cells, NK-92 had cytotoxicity of81 to 47%, NK-92MI had cytotoxicity of 75 to 65% and NK-92CI hadcytotoxicity of 82 to 52%.

Example 18

[0141] Effect of Transfected NK-92 Cells on Hematopoietic ProgenitorCells. One potential clinical application of the NK-92, NK-92MI andNK-92CI cells is as an ex vivo purging agent for autologous grafts. Inorder for the NK cells to be suitable for such a purpose, they must beable to purge the malignant cells without killing the hematopoieticprogenitor cells in the graft or influencing their hematopoieticpotential. In order to assay this, a colony-forming cell assay (CFC) wasperformed where the clonogenic output of PBMCs was examined following a48 hour incubation with NK-92MI and NK-92CI at various E:T ratios. NK-92was previously shown to have minimal effect on hematopoietic stem cells(Example 6). In this example, NK-92MI and NK-92CI also show little or noeffect on clonogenic output The number of total colonies followingincubation with either NK-92MI or NK-92CI was very similar to control,although a slight decrease was seen with the highest effector:PBMC ratioof 1:1 (FIG. 19). Total clonogenic output from both NK-92MI and NK-92CIwas approximately 80% of control under this condition. However, noconsistent trend was seen in terms of clonogenic output with respect tothe ratio of NK:PBMCs. In terms of specific colony types, there were nodetectable differences in the number of output BFU-E colonies, which arethe most numerous. Some effect was seen with both the CFU-GM andCFU-GEMM colonies. However, the absolute numbers of these colonies arevery low, making any conclusions difficult since small variations in thenumber of colonies has a large effect on the calculation of clonogenicoutput. An influence on CFU-GM and CFU-GEMM is seen at higher ratios,but no consistent correlation between ratio and output was noted.

Example 19

[0142] Irradiation of the Transfected NK-92 Cells. To establish aneffective irradiation dose to inhibit proliferation and maintaincytotoxicity, NK-92MI and NK-92CI cells were irradiated at 500, 1,000,1,500 and 2,000 cGy and assayed for proliferation by the ³H thymidineincorporation assay (see Examples 7 and 8). Both NK-92MI and NK-92CIwere more sensitive to irradiation than the parental NK-92 cell.Proliferation of NK-92MI and NK-92CI was found to be more stronglysuppressed than NK-92 at all radiation doses tested (FIG. 20, Panel A).For NK-92MI and NK-92CI, proliferation was completely suppressed by aradiation dose between 500 and 1,000 cGy. The level of thymidineincorporation reached a plateau at approximately 20% of unirradiatedcontrol cells for NK-92CI and 10% for NK-92MI. For determination ofviability, NK-92, NK-92MI and NK-92CI cells were irradiated at 250, 500,1,000 and 2,000 cGy and trypan blue exclusion was determined 24, 48 and72 hours following irradiation. It was found that greater percentages ofboth NK-92MI and NK-92CI were found to be killed by irradiation ascompared to the parental cells at equivalent doses (FIG. 20, Panel B).Viability of NK-92 was higher than that of both transfectants at alldose rates tested.

[0143] The cytotoxicity of these cells following irradiation is shown inFIG. 21. Cells irradiated at 0, 1,000 and 2,000 cGy were tested afterthree days for cytotoxicity against K562 and Raji cells ateffector:target ratios of 20:1, 10:1, 5:1 and 1:1. Cytotoxicity of NK-92cells three days following irradiation at 1,000 cGy was determined to beapproximately 10-30% K562 (FIG. 21, Panel A) and 30-50% and for Raji(FIG. 21, Panel B). Irradiation at 2,000 cGy resulted in cytotoxicity of1-5% against K562 and 3-13% against Raji. In contrast, NK-92MI had only0-5% and 0-1% cytotoxic activity against K562 and 0-1% and 0% againstRaji three days after irradiation doses of 1,000 and 2,000 cGy,respectively. NK-92CI had only 1-4% cytotoxicity to K562 and 2-7% toRaji three days after irradiation at 1000 cGy and 0% to K562 and 0-2%after irradiation with 2000 cGy.

[0144] In the data reported here, IL-2 transfectants are seen to be moresensitive to irradiation than the parental strain. Proliferation andcytotoxicity of both NK-92MI and NK-92CI cells were suppressed at alower radiation level than for-the parental strains, andradiation-induced lethality was much greater in the IL-2-independentmodified cells in NK-92 at equivalent radiation doses. The highIL-2-producing NK-92MI is more sensitive to radiation than the low IL-2producing NK-92CI variant. As a result of the increased radiationsensitivity, a reduced level of irradiation would be sufficient toadequately control proliferation while minimizing lethality to the cellsand inhibition of cytotoxicity. In routine experiments, the worker ofordinary skill would be able to repeat experiments such as thosedescribed in this example. By using lower radiation doses, in the rangebetween 0 and 1000 cGy optimal doses can be determined that inhibitproliferation while maintaining viability and cytolytic activity inNK-92MI and NK-92CI.

Example 20

[0145] Transfection of NK-92 with a gene for thymidine kinase. NK-92cells are to be transfected with a vector bearing a gene for thymidinekinase (TK). The resulting TK-modified NK-92 cells are thereby renderedsusceptible to the toxic effects of the guanosine analogs, gancyclovir,and acyclovir.

[0146] A vector suitable for transfecting a mammalian cell is to beconstructed, such as a retroviral vector harboring a herpes simplexvirus (HSV) TK gene, under the control of the HSV TK promoter, andcontaining its own polyA addition site. Transfection is to be carriedout by a method known to those skilled in cell biology and mammalianmolecular biology, such as by electroporation (Bio-Rad Gene Pulser™), orby lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413(1987)). The transfected NK-92 cells so produced are susceptible toinactivation by administering gancyclovir or acyclovir.

Example 21

[0147] Mutation of NK-92 HLA cell surface protein. NK-92 cells are to beobtained from the cell line described by Gong et al. (1994). Thechromosome bearing the β₂-microglobulin gene is to be isolated, and theDNA contained within this chromosome is to be purified away fromhistones and other DNA-bound proteins. The gene fragment bearingβ₂-microglobulin is to be excised with restriction nucleases, and sitespecific mutagenesis is to be conducted via an oligonucleotide cassetteharboring the mutated nucleotide sequence. These procedures employtechniques commonly known in recombinant DNA technology, as set forth,for example, in “Current Protocols in Molecular Biology”, Ausubel etal., John Wiley and Sons, New York 1987 (updated quarterly), and“Molecular Cloning: A Laboratory Manual”, 2nd Ed., Sambrook, Fritsch andManiatis, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989,incorporated herein by reference. The mutated β₂-microglobulin is to bereincorporated into the cellular DNA, and reintroduced into the NK-92cells. This preparation of cells will then express cell surface HLAmolecules incorporating mutated β₂-microglobulin moieties, and will havelost the ability to bind T-cell receptors.

Example 22

[0148] NK-92 Cells Expressina Receptors for a Cancer Cell. The CTL of apatient suffering from a cancer are to be harvested by differentialcentrifugation on a density gradient. The CTL are to be immunoaffinitypurified to contain predominantly the CTL targeting a receptor on thecancer cell from the patient. The DNA of the CTL population obtained isto be isolated, and the genes for the MHC class I receptor in thecancer-targeted CTL isolated by restriction nuclease cleavage. The genesso purified are to be amplified using the polymerase chain reaction, andthe resulting amplified genes incorporated into a vector suitable forthe constitutive expression of the genes in NK-92 cells. The vectors areto be transfected into NK-92 cells, and the modified NK-92 cells soobtained are to be selected using, for example, an antibiotic resistancemarker incorporated into the vector. The cells so selected are to becultured to increase their number. They may then be employed to targetspecifically the cancer cells in the patient, and treat the canceroccurring in the patient either ex vivo or in vivo.

Example 23

[0149] Use of NK-92 Cells to Kill HIV-Infected Cells. 8E5 is a cell lineharboring HIV that produces HIV virions. 8E5L is a corresponding cellline infected with HIV which does not produce virions. In a cytotoxicactivity experiment in which the chromium release assay was used toevaluate activity, the results presented in Table 8 were obtained. Inthese experiments, A3.01 cells are an uninfected control cell line.TABLE 8 Cytotoxic Activity of NK-92 Cells on HIV-Infected Cells. TargetE:T Ratio % Cytotoxicity A3.01 50:1 43 20:1 51  5:1 44  1:1 44 8E5L 50:143 20:1 37  5:1 44  1:1 40 8E5 50:1 76 20:1 69  5:1 77  1:1 65

[0150] It is seen from Table 8 that 8E5 cells which produce HIVparticles elicit a higher cytotoxic activity than do 8E5L cells, whichdo not produce HIV particles, and higher than control cells. Withoutwishing to be bound by theory, it is believed that the anti-viral effectof NK-92 cells is due to factors such as a direct cytotoxic effect, aswell as inhibition through MIP-1α, which is produced by NK-92 cells inhigh concentrations (Bluman et al., J. Clin. Investig. 97, 2722 (1996)).The results indicate that NK-92 cells effectively lyse HIV-producingcells in vitro.

1 2 1 20 DNA Artificial sequence primer oligonucleotide based on humansequence 1 caactcctgt cttgcattgc 20 2 19 DNA Artificial sequence primeroligonucleotide based on human sequence 2 gcatcctggt gagtttggg 19

That which we claim is:
 1. A method of purging cells related to apathology from a biological sample, said method comprising (i) obtaininga biological sample from a mammal, wherein the biological sample issuspected of containing cells related to the pathology; and (ii)contacting the biological sample with a medium comprising NK-92 ormodified NK-92 natural killer cells, wherein the modified NK-92 cellshave been modified by a physical treatment or by transfection with avector; whereby the natural killer cells purge cells related to thepathology from the sample.
 2. The method described in claim 1 whereinthe pathology is a cancer.
 3. The method described in claim 1 whereinthe pathology is an infection by a pathogenic virus.
 4. The methoddescribed in claim 3 wherein the pathogenic virus is humanimmunodeficiency virus, Epstein-Barr virus, cytomegalovirus, or herpesvirus.
 5. The method described in claim 1 wherein the biological sampleis human blood or bone marrow.
 6. The method described in claim 1wherein the natural killer cell is immobilized on a support.
 7. Themethod described in claim 1 wherein the modified NK-92 cells have beenmodified by a physical treatment that renders them non-proliferative,said treatment not significantly diminishing their cytotoxicity, bytreatment that inhibits expression of HLA antigens on the NK-92 cellsurface, by transfection with a vector, or by any combination thereof.8. The method described in claim 7 wherein the cells have beentransfected with a vector encoding a cytokine that promotes the growthof the cells, a vector encoding a protein that is responsive to anagent, a vector encoding a cancer cell receptor molecule, or with anycombination thereof.
 9. The method described in claim 1 wherein themedium further comprises cytokine that promotes the growth of the cells.10. A method of treating a pathology ex vivo in a mammal comprising thesteps of: (i) obtaining a biological sample from the mammal, wherein thesample is suspected of containing cells related to the pathology; (ii)contacting the biological sample with a medium comprising NK-92 ormodified NK-92 natural killer cells, wherein the modified NK-92 cellshave been modified by a physical treatment or by transfection with avector, whereby the cells related to the pathology in the sample areselectively destroyed, thereby producing a purged sample; and (iii)returning the purged sample to the mammal.
 11. The method described inclaim 10 wherein the pathology is a cancer.
 12. The method described inclaim 11 wherein the cancer is a leukemia, a lymphoma or a multiplemyeloma.
 13. The method described in claim 10 wherein the pathology isan infection by a pathogenic virus.
 14. The method described in claim 13wherein the pathogenic virus is human immunodeficiency virus,Epstein-Barr virus, cytomegalovirus, or herpes virus.
 15. The methoddescribed in claim 10 wherein the biological sample is blood or bonemarrow and wherein the mammal is a human.
 16. The method described inclaim 10 wherein the natural killer cell is immobilized on a support.17. The method described in claim 10 wherein the medium comprisesmodified NK-92 cells which have been modified by a physical treatmentthat renders them non-proliferative, said treatment not significantlydiminishing their cytotoxicity, by treatment that inhibits expression ofHLA antigens on the NK-92 cell surface, by transfection with a vector,or by any combination thereof.
 18. The method described in claim 17wherein the cells have been transfected with a vector encoding acytokine that promotes the growth of the cells, a vector encoding aprotein that is responsive to an agent, a vector encoding a cancer cellreceptor molecule, or with any combination thereof.
 19. The method oftreating a cancer described in claim 10 wherein the medium furthercomprises a cytokine that promotes the growth of the cells.
 20. A methodof treating a pathology in vivo in a mammal comprising the step ofadministering to the mammal a medium comprising NK-92 or modified NK-92natural killer cells, wherein the modified NK-92 cells have beenmodified by a physical treatment that renders them non-proliferative,said treatment not significantly diminishing their cytotoxicity, bytreatment that inhibits expression of HLA antigens on the NK-92 cellsurface, by transfection with a vector, or by any combination thereof.21. The method described in claim 20 wherein the modified NK-92 cellshave been transfected with a vector encoding a cytokine that promotesthe growth of the cells, with a vector encoding a protein that isresponsive to an agent, a vector encoding a cancer cell receptormolecule, or with any combination thereof.
 22. The method described inclaim 20 wherein the pathology is a cancer.
 23. The method of treating acancer described in claim 22 wherein the cancer is a leukemia, alymphoma or a multiple myeloma.
 24. The method described in claim 20wherein the pathology is an infection by a pathogenic virus.
 25. Themethod described in claim 24 wherein the pathogenic virus is humanimmunodeficiency virus, Epstein-Barr virus, cytomegalovirus, or herpesvirus.
 26. The method of treating a pathology described in claim 20wherein the route of administration of the cells to the mammal isintravenous and the mammal is a human.
 27. The method of treating apathology described in claim 20 further comprising administering acytokine that promotes the growth of the cells to the mammal inconjunction with administering the medium comprising the natural killercell.
 28. The method of treating a pathology described in claim 26wherein the NK-92 is modified by transfection with a vector encoding aprotein that is responsive to an agent such that when the agent is takenup by the cell, the cell is inactivated, and wherein the method furthercomprises administering to the mammal, after a time sufficient for thenatural killer cell to treat the cancer has elapsed, an amount of theagent effective to inactivate the cell.
 29. The method of treating apathology described in claim 28 wherein the agent is acyclovir organcyclovir.